Ageing and longevity are related to growth hormone/insulin-like growth factor-1 secretion

BACKGROUND: It is known that the growth process is related to an individual's life-span, but the role of growth hormone (GH) secretion in human ageing remains unknown. OBJECTIVE: This study has focussed on the influence of GH on ageing parameters and on its relationship with human longevity. METHODS: To deal with the first issue, we compared ageing parameters of young (up to 39) and old (over 70) individuals having similar insulin-like growth factor-1 (IGF-1) blood levels. For the second one, the decline in IGF-1 levels was studied comparing its behaviour in the first half with that in the second half of adult life. The latter represents the period of life in which mortality progressively increases. Two hundred and five healthy individuals were chosen as subjects, well distributed by gender and age (between 19 and 93 years). RESULTS: Old males with IGF-1 levels similar to young ones do not show the age-dependent decrease in serum testosterone and lean body mass, nor the increase in fat body mass. Other hormone-metabolic and nutritional parameters do not reveal any change compared with the results of all individuals. In females, the results do not allow to assume any IGF-1 influence. The behaviour of the linear regression in the second half of adult life of males, which becomes flat because old men having low IGF-1 blood levels die earlier, is consistent with these results. This effect, which is supported by predictive analysis, is not observed in females, i.e. the IGF-1 level declines in the second half of the women's adult life are only a little flatter than in the first half. Finally, extrapolating the regressions obtained in the first half of adulthood, the age at which the curve crosses the x-axis is 110 years for males and 132 for females. CONCLUSIONS: The presented study of IGF-1 levels suggests that the GH secretion in adulthood plays a determinant role not only for some regressive manifestations, but also for life potential.     

Cardiovascular action of insulin-like growth factor-1 is not mediated by calcitonin gene-related peptide neurons

Calcitonin gene-related peptide (CGRP) is a potent vasodilator located in the peripheral nerves including the perivascular nerves. Previous studies in our laboratory determined that the vasodilatory action of insulin is mediated in part by CGRP-containing neurons. Since insulin-like growth factor-1 (IGF-1) and insulin share molecular and receptor structural similarity as well as functional similarity, we investigated the role of the CGRP-containing neurons in IGF-1-mediated vasodilation. Wistar rats were made CGRP deficient by treatment with capsaicin (50 mg/kg) 1-3 d after birth. Vehicle-treated controls and CGRP-deficient rats were maintained for 12 to 13 wk. At this time rats were fasted overnight, anesthetized with urethane and chloralose, and prepared for cardiovascular recordings. The basal mean arterial pressure (MAP) was higher in CGRP-deficient rats when compared with controls. The infusion of IGF-1 resulted in an equivalent decrease in MAP in both the CGRP-deficient and control rats. IGF-1 infusion did not change the heart rate in control rats but decreased it in CGRP-deficient rats. IGF-1 also increased flow as determined by conductance in the iliac, renal, and superior mesenteric vascular beds in both vehicle controls and CGRP-deficient rats. We concluded that unlike insulin the IGF-1-mediated vasodilatory response is not mediated by the CGRP-dependent perivascular neurons.     

Insulin but not insulin-like growth factor-1 promotes the primordial to primary follicle transition

A critical step in ovarian biology is the transition of the developmentally arrested primordial follicle to the growing primary follicle. The current study utilizes a rat ovarian organ culture system to investigate the role of insulin and insulin-like growth factor-1 (IGF-1) in this process. Four-day-old rat ovaries were cultured and the degree of primordial to primary follicle transition measured. Insulin increased the primordial to primary follicle transition 30% over control with a half maximal effective concentration (EC50) between 2.5 and 5 ng/ml. IGF-1 did not cause an increase in the primordial to primary follicle transition at concentrations up to 100 ng/ml. Ovaries were also treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) and neither had an effect on the primordial to primary follicle transition. Ovaries were treated with insulin in conjunction with other factors known to promote the primordial to primary follicle transition in order to discern any potential synergistic effects. Previous experiments have shown that kit ligand (KL), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) promote the primordial to primary follicle transition. Insulin was shown to have an additive effect with KL and LIF, but not bFGF. The fact that insulin can influence the primordial to primary follicle transition at low concentrations (i.e. 5 ng/ml) and that IGF-1 has no effect suggests that insulin is acting at the insulin receptor, not the IGF-1 receptor. The observation that insulin has an additive effect with KL and LIF, but not bFGF, suggests the insulin's site of action is likely the oocyte. In summary, observations suggest that insulin acts as an endocrine type factor to help coordinate primordial to primary follicle transition at the level of the oocyte. The significance of the observations in relation to diabetes and female infertility is discussed.     

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.     

Pentagastrin gastroprotection against acid is related to H2 receptor activation but not acid secretion

Background—Pentagastrin enhancesgastric mucosal defence mechanisms against acid and protects thegastric mucosa from experimental injury.
Aims—To investigate whether thisgastroprotection is mediated by histamine receptors or occurs as asecondary effect of acid secretion stimulation.
Methods—The effects ofomeprazole (100 µmol/kg), ranitidine (20 mg/kg), and pyrilamine (10 mg/kg) on pentagastrin (80 µg/kg/h) induced gastroprotection againstacidified aspirin injury were examined in a luminal pH controlledmodel. The effects of these compounds on pentagastrin enhancedgastroprotective mechanisms were investigated using intravitalmicroscopy, in which intracellular pH of gastric surface cells(pH_i), mucus gel thickness, gastric mucosal blood flow, andacid output were measured simultaneously.
Results—Pentagastrin protected ratgastric mucosa from acidified aspirin injury. This gastroprotection wasabolished by ranitidine, but not omeprazole or pyrilamine. Pentagastrininduced a hyperaemic response to luminal acid challenge, increasedmucus gel thickness, and elevated pH_i during acidchallenge. Ranitidine reversed these enhanced defence mechanisms,whereas omeprazole and pyrilamine preserved these effects.
Conclusions—These dataindicate that pentagastrin associated gastroprotection and enhanceddefence mechanisms against acid result mainly from activation ofhistamine H₂ receptors, and not as an effect of thestimulation of acid secretion.

Keywords:gastric injury; gastric defence mechanisms; omeprazole; pyrilamine; ranitidine; intracellular pH

     

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.     

Increased, not decreased activation of the insulin-like growth factor (IGF) receptor signalling pathway during ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro. The IGF-I receptor (IGF-IR) is believed to mediate protective effects of the IGFs against apoptosis. To determine whether ceramide-mediated induction of apoptosis involved a decreased survival effect of the IGF-IR, apoptosis was induced in IGF-I receptor positive (R+) and negative (R-) murine fibroblasts by incubation with increasing doses of the sphingolipid analogue, C2 ceramide. Lower ceramide doses were required to induce death in receptor negative compared with receptor positive fibroblasts (P< 0.05 at ceramide doses of 2 microM or greater), not only corroborating evidence that the IGF-I receptor functions as a survival receptor, but also suggesting that ceramide is not inducing apoptosis by suppressing a survival effect of the IGF-IR. Ceramide has been reported to induce death through suppression of MAP kinase, and activation of JUN kinase signalling; since our initial data suggested that ceramide had not affected an anti-apoptotic signalling event of the IGF-IR, we monitored the activation of these enzymes. To our surprise, in the presence of ceramide, not only was JUN kinase activity increased, but so too was MAP kinase. Inhibition of MAP kinase, using the MEKK inhibitor, PD98059, significantly reduced ceramide-induced cell death (P< 0. 001). Ceramide also enhanced IGF-induced tyrosine phosphorylation of the IGF-I receptor and activated PI-3 kinase. The cumulative effects of these events resulted in increased progression to the G2 phase of the cell cycle, arrest without subsequent mitosis, and apoptosis. These results indicate that ceramide is capable of eliciting apparently contradictory events within a single cell type, and suggest that in the presence of an IGF-IR, survival is enhanced because ceramide can activate PI-3 kinase, believed to be an anti-apoptotic enzyme.     

Combined exercise and insulin-like growth factor-1 supplementation induces neurogenesis in old rats, but do not attenuate age-associated DNA damage

We have investigated the effects of 2 weeks of insulin-like growth factor-1 (IGF-1) supplementation (5 μg/kg per day) and 6 weeks of exercise training (60% of the maximal oxygen consumption [VO? max]) on neurogenesis, DNA damage/repair, and sirtuin content in the hippocampus of young (3 months old) and old (26 months old) rats. Exercise improved the spatial memory of the old group, but IGF-1 supplementation eliminated this effect. An age-associated decrease in neurogenesis was attenuated by exercise and IGF-1 treatment. Aging increased the levels of 8-oxo-7,8-dihydroguanine (8-oxoG) and the protein Ku70, indicating the role of DNA damage in age-related neuropathology. Acetylation of 8-oxoguanine DNA glycosylase (OGG1) was detected in vivo, and this decreased with aging. However, in young animals, exercise and IGF-1 treatment increased acetylated (ac) OGG1 levels. Sirtuin 1 (SIRT1) and SIRT3, as DNA damage-associated lysine deacetylases, were measured, and SIRT1 decreased with aging, resulting in a large increase in acetylated lysine residues in the hippocampus. On the other hand, SIRT3 increased with aging. Exercise-induced neurogenesis might not be a causative factor of increased spatial memory, because IGF-1 plus exercise can induce neurogenesis in the hippocampus of older rats. Data revealed that the age-associated increase in 8-oxoG levels is due to decreased acetylation of OGG1. Age-associated decreases in SIRT1 and the associated increase in lysine acetylation, in the hippocampus, could have significant impact on function and thus, could suggest a therapeutic target.     

Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice.

Transgenic mice were generated in which the cDNA for the human insulin-like growth factor 1B (IGF-1B) was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene, IGF-1B mRNA was not detectable in the fetal heart at the end of gestation, was present in modest levels at 1 day after birth, and increased progressively with postnatal maturation, reaching a peak at 75 days. Myocytes isolated from transgenic mice secreted 1.15 +/- 0.25 ng of IGF-1 per 10(6) cells per 24 hr versus 0.27 +/- 0.10 ng in myocytes from homozygous wild-type littermates. The plasma level of IGF-1 increased 84% in transgenic mice. Heart weight was comparable in wild-type littermates and transgenic mice up to 45 days of age, but a 42%, 45%, 62%, and 51% increase was found at 75, 135, 210, and 300 days, respectively, after birth. At 45, 75, and 210 days, the number of myocytes in the heart was 21%, 31%, and 55% higher, respectively, in transgenic animals. In contrast, myocyte cell volume was comparable in transgenic and control mice at all ages. In conclusion, overexpression of IGF-1 in myocytes leads to cardiomegaly mediated by an increased number of cells in the heart.     

Caveolin-1 down-regulation inhibits insulin-like growth factor-I receptor signal transduction in H9C2 rat cardiomyoblasts

Caveolin (Cav)-1, the major caveolar protein, directly interacts with IGF-I receptor (IGF-IR) and its intracellular substrates. To determine the role of Cav-1 in IGF-IR signaling, we transfected H9C2 cells with small interfering RNA specific for Cav-1-siRNA. The selective down-regulation of Cav-1 (90%) was associated with a smaller reduction of Cav-2, whereas Cav-3 expression was unaffected. A significant reduction of IGF-IR tyrosine phosphorylation in Cav-1-siRNA H9C2 cells was found compared with H9C2 control cells (Ctr-siRNA). The reduced IGF-IR autophosphorylation resulted in a decrease of insulin receptor substrate-1, Shc, and Akt activation. In addition, in Cav-1-siRNA H9C2 cells, IGF-I did not prevent apoptosis, suggesting that Cav-1 is required to mediate the antiapoptotic effect of IGF-I in cardiomyoblasts. The down-regulation of Cav-1 decreased IGF-IR activation and affected the ability of IGF-I to prevent apoptosis after serum withdrawal also in human umbilical vein endothelial cells. These results demonstrate that: 1) Cav-1 down-regulation negatively affects IGF-IR tyrosine phosphorylation; 2) this effect causes a reduced activation of insulin receptor substrate-1, Shc, and Akt; and 3) Cav-1 is involved in IGF-IR antiapoptotic signaling after serum deprivation.     

Increased expression of the insulin-like growth factor I receptor gene, IGF1R, in Wilms tumor is correlated with modulation of IGF1R promoter activity by the WT1 Wilms tumor gene product.

Wilms tumor is a pediatric neoplasm that arises from the metanephric blastema. The expression of the gene encoding insulin-like growth factor II (IGF-II) is often elevated in these tumors. Since many of the actions of IGF-II are mediated through activation of the IGF-I receptor (IGF-IR), we have measured the levels of IGF-IR mRNA in normal kidney and in Wilms tumor samples using solution hybridization/RNase protection assays. IGF-IR mRNA levels in the tumors were 5.8-fold higher than in adjacent normal kidney tissue. Among the tumors themselves, the levels of IGF-IR mRNA in those containing heterologous stromal elements were 2-fold higher (P < 0.01) than in tumors without these elements. IGF-IR gene (designated IGF1R) expression in the tumors was inversely correlated with the expression of the Wilms tumor suppressor gene WT1, whose inactivation appears to be a key step in the etiology of Wilms tumor. Cotransfection of Chinese hamster ovary cells with rat and human IGF-IR gene promoter constructs driving luciferase reporter genes and with WT1 expression vectors showed that the active WT1 gene product represses IGF-IR promoter activity in a dose-dependent manner. These results suggest that underexpression, deletion, or mutation of WT1 may result in increased expression of the IGF-IR, whose activation by IGF-II may be an important aspect of the biology of Wilms tumor.     

Glycogen synthase kinase-3beta is involved in the process of myocardial hypertrophy stimulated by insulin-like growth factor-1.

BACKGROUND: Glycogen synthase kinase-3 beta (GSK-3beta) is involved in many cellular processes, such as metabolism, apoptosis, differentiation and proliferation. Insulin-like growth factor-1 (IGF-1), which is well known to have a hypertrophic effect on cardiomyocytes, inactivates (phosphorylates) GSK-3beta in some cell types. The role of GSK-3beta in cardiomyocytes as a negative regulator of cardiac hypertrophy has been recently reported and the present study investigated the role of GSK-3beta in the cardiac hypertrophy of cultivated neonatal rat cardiomyocytes induced by IGF-1. METHODS AND RESULTS: First, the IGF-1 induced signal transduction leading to GSK-3beta in neonatal rat cardiomyocytes was examined. The phosphatidylinositol (PI) 3-kinase/Akt/GSK-3 beta signaling induced by IGF-1 was investigated using inhibitors of PI 3-kinase and Ad AktAA, a dominant negative form of Akt. Furthermore, using Ad MEK DN, a dominant negative form of MEK, it was found that MEK negatively regulates Akt phosphorylation upon IGF-1 stimulation. Next, it was examined whether GSK-3beta acts as a negative regulator in the cardiac hypertrophy induced by IGF-1. Sustained stimulation by IGF-1 caused cardiac hypertrophy in protein synthesis and cellular morphology, and overexpression of unphosphorylatable GSK-3beta (Ad GSK-3beta S9A) repressed these hypertrophic effects of IGF-1. CONCLUSIONS: GSK-3beta may play an important role as a negative regulator of cardiac hypertrophy induced by IGF-1.     

The chick embryo fibroblast cation-independent mannose 6-phosphate receptor is functional and immunologically related to the mammalian insulin-like growth factor-II (IGF-II)/man 6-P receptor but does not bind IGF-II.

The insulin-like growth factor-II (IGF-II)/Mannose 6-P receptor (Man 6-P) is a multifunctional receptor that binds two unrelated ligands, IGF-II and lysosomal enzymes that contain Man 6-P recognition markers. Although this receptor has been extensively characterized in mammalian cells, binding of radiolabeled IGF-II to this receptor in avian cells and tissues has not been reported. In the present study, we demonstrate that chick embryo fibroblasts (CEFs) bind and internalize lysosomal enzymes in a Man 6-P-inhibitable fashion, and possess a protein immunologically related to the mammalian IGF-II/Man 6-P receptor that binds lysosomal enzymes with Man 6-P recognition markers but does not bind IGF-II. 1) When lysates of biosynthetically labeled CEFs were affinity-purified on beta-galactosidase-Sepharose, an approximately 250 kilodalton protein was observed in the Man 6-P eluate but not in the Glc 1-P or mannose eluates, that was precipitated by antisera to purified rat and bovine IGF-II/Man 6-P receptors, but not by nonimmune serum. 2) When CEFs were incubated with [35S]proteins enriched in lysosomal enzymes, Man 6-P inhibited binding (0 C) and uptake (34 C) in a dose-dependent fashion. Binding was unchanged in the absence of divalent cations. At low sugar concentrations, binding and uptake were inhibited selectively by Man 6-P and the conformationally similar sugar phosphate, Fru 1-P, a specificity similar to that of mammalian cation-independent Man 6-P receptors. 3) When affinity-purified lysates from biosynthetically labeled CEFs were incubated with antiserum to the rat IGF-II/Man 6-P receptor, a 245 kilodalton protein was immunoprecipitated from lysates that had been affinity purified on beta-galactosidase-Sepharose but not after purification on IGF-II-Sepharose. By contrast, a truncated IGF-II/Man 6-P receptor, presumably internalized from the fetal bovine serum used to feed the cells, was purified from lysates of unlabeled CEFs on IGF-II-Sepharose. Thus, CEFs possess a cation-independent Man 6-P receptor that is similar in size and immunological reactivity to the mammalian IGF-II/Man 6-P receptor, and binds and internalizes lysosomal enzymes but, unlike the mammalian receptor, does not bind IGF-II.