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1.  Stress resistance and aging: Influence of genes and nutrition 
Previous studies have shown that dermal fibroblast cell lines derived from young adult mice of the long-lived Snell dwarf (dw/dw), Ames dwarf (df/df) and growth hormone receptor knockout (GHR-KO) mouse stocks are resistant, in vitro, to the cytotoxic effects of hydrogen peroxide, cadmium, ultraviolet light, paraquat, and heat. Here we show that, in contrast, fibroblasts from mice on low-calorie (CR) or low methionine (Meth-R) diets are not stress resistant in culture, despite the longevity induced by both dietary regimes. A second approach, involving induction of liver cell death in live animals using acetaminophen (APAP), documented hepatotoxin resistance in the CR and Meth-R mice, but dw/dw and GHR-KO mutant mice were not resistant to this agent, and were in fact more susceptible than littermate controls to the toxic effects of APAP. These data thus suggest that while resistance to stress is a common characteristic of experimental life span extension in mice, the cell types showing resistance may differ among the various models of delayed or decelerated aging.
doi:10.1016/j.mad.2006.04.002
PMCID: PMC2923407  PMID: 16713617
Stress resistance; Caloric restriction; Methionine restriction; Snell dwarf; Growth hormone receptor knockout
2.  Upregulation of the Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Receptor Axis in the Heart and the Kidney of Growth Hormone Receptor knock-out Mice 
Objective
Growth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR−/−) mouse.
Design
RAS components were evaluated in the heart and the kidney of GHR−/− and control mice by immunohistochemistry and western blotting (n=12 for both groups).
Results
The immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR−/− mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR−/− mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western Blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues.
Conclusions
The shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR−/− mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.
doi:10.1016/j.ghir.2012.08.003
PMCID: PMC3698955  PMID: 22947377
Angiotensin-(1-7); AT1 receptor; Mas receptor; Growth hormone; Renin-angiotensin system
3.  THE GROWTH HORMONE RECEPTOR (GHR) POLYMORPHISM IN GROWTH-RETARDED CHILDREN WITH CUSHING DISEASE: LACK OF ASSOCIATION WITH GROWTH AND MEASURES OF THE SOMATOTROPIC AXIS 
Objective
Pediatric Cushing disease (CD) often presents with short stature but we have observed significant inter-individual variability in the growth delay caused by endogenous hypercortisolism. Glucocorticoids cause growth retardation by affecting the growth hormone (GH) – insulin-like growth factor-1 (IGF 1) somatotropic axis, but also other, GH-independent sites. Recently, the GH receptor (GHR) gene was found to have a common polymorphism (P) that leads to a deletion (d3) or retention of exon 3. In this study, we tested the hypothesis that the GH receptor polymorphism (GHR-P) maybe one of the significant variants that determine the degree of growth delay among patients with CD.
Design and methods
GHR genotyping was performed on 56 children with newly diagnosed CD (24 females, 32 males, mean age of 12.9±3.3 years) who were followed at our institution between the years 1997–2007. Correlation analysis included genotype, measures of growth and the somatotropic axis, and anthropometrics.
Results
Within the group, 31 (12 girls, 19 boys) expressed the full length GHR allele, 10 (4 girls, 6 boys) were d3-GHR homozygotes and 15 (7 girls, 8 boys) were d3-GHR heterozygotes. No significant differences were found between the GHR genotypes and patient’s height and or growth velocity, or any other measures that we evaluated.
Conclusions
The presence of a well-studied and common GHR polymorphism does not appear to be responsible for the variability of growth delay observed in patients with Cushing disease.
doi:10.1055/s-0029-1242744
PMCID: PMC3412355  PMID: 20013551
Cushing syndrome; cortisol; pituitary gland; growth hormone receptor; genetics
4.  Metabolic characteristics of long-lived mice 
Frontiers in Genetics  2012;3:288.
Genetic suppression of insulin/insulin-like growth factor signaling (IIS) can extend longevity in worms, insects, and mammals. In laboratory mice, mutations with the greatest, most consistent, and best documented positive impact on lifespan are those that disrupt growth hormone (GH) release or actions. These mutations lead to major alterations in IIS but also have a variety of effects that are not directly related to the actions of insulin or insulin-like growth factor I. Long-lived GH-resistant GHR-KO mice with targeted disruption of the GH receptor gene, as well as Ames dwarf (Prop1df) and Snell dwarf (Pit1dw) mice lacking GH (along with prolactin and TSH), are diminutive in size and have major alterations in body composition and metabolic parameters including increased subcutaneous adiposity, increased relative brain weight, small liver, hypoinsulinemia, mild hypoglycemia, increased adiponectin levels and insulin sensitivity, and reduced serum lipids. Body temperature is reduced in Ames, Snell, and female GHR-KO mice. Indirect calorimetry revealed that both Ames dwarf and GHR-KO mice utilize more oxygen per gram (g) of body weight than sex- and age-matched normal animals from the same strain. They also have reduced respiratory quotient, implying greater reliance on fats, as opposed to carbohydrates, as an energy source. Differences in oxygen consumption (VO2) were seen in animals fed or fasted during the measurements as well as in animals that had been exposed to 30% calorie restriction or every-other-day feeding. However, at the thermoneutral temperature of 30°C, VO2 did not differ between GHR-KO and normal mice. Thus, the increased metabolic rate of the GHR-KO mice, at a standard animal room temperature of 23°C, is apparently related to increased energy demands for thermoregulation in these diminutive animals. We suspect that increased oxidative metabolism combined with enhanced fatty acid oxidation contribute to the extended longevity of GHR-KO mice.
doi:10.3389/fgene.2012.00288
PMCID: PMC3521393  PMID: 23248643
growth hormone; aging; calorie restriction; dwarf mice; metabolism
5.  Growth hormone-releasing hormone disruption extends lifespan and regulates response to caloric restriction in mice 
eLife  2013;2:e01098.
We examine the impact of targeted disruption of growth hormone-releasing hormone (GHRH) in mice on longevity and the putative mechanisms of delayed aging. GHRH knockout mice are remarkably long-lived, exhibiting major shifts in the expression of genes related to xenobiotic detoxification, stress resistance, and insulin signaling. These mutant mice also have increased adiponectin levels and alterations in glucose homeostasis consistent with the removal of the counter-insulin effects of growth hormone. While these effects overlap with those of caloric restriction, we show that the effects of caloric restriction (CR) and the GHRH mutation are additive, with lifespan of GHRH-KO mutants further increased by CR. We conclude that GHRH-KO mice feature perturbations in a network of signaling pathways related to stress resistance, metabolic control and inflammation, and therefore provide a new model that can be used to explore links between GHRH repression, downregulation of the somatotropic axis, and extended longevity.
DOI: http://dx.doi.org/10.7554/eLife.01098.001
eLife digest
There is increasing evidence that the hormonal systems involved in growth, the metabolism of glucose, and the processes that balance energy intake and expenditure might also be involved in the aging process. In rodents, mutations in genes involved in these hormone-signaling pathways can substantially increase lifespan, as can a diet that is low in calories but which avoids malnutrition. As well as living longer, such mice also show reductions in age-related conditions such as diabetes, memory loss and cancer.
Many of these effects appear to involve the actions of growth hormone. Mice with mutations that disrupt the development of the pituitary gland, which produces growth hormone, show increased longevity, as do mice that lack the receptor for growth hormone. However, these animals also show changes in a number of other hormones, making it difficult to be sure that the reduction in growth hormone signaling is responsible for their increased lifespan.
Now, Sun et al. have studied mutant mice that lack a gene called GHRH, which promotes the release of growth hormone. These mice, which have normal levels of all other pituitary hormones, lived for up to 50% longer than their wild-type littermates. They were more active than normal mice and had more body fat, and showed greatly increased sensitivity to insulin.
Some of the changes in these mutant mice resembled those seen in animals with a restricted calorie intake, suggesting that the same mechanisms may be implicated in both. However, Sun et al. found that caloric restriction further increased the lifespans of their GHRH knockout mice, indicating that at least some of the effects of caloric restriction are independent of disrupted growth hormone signaling.
The results of this study are an important step forward for understanding how growth hormone signaling and caloric restriction regulate aging, both individually and in combination. The GHRH knockout mice are likely to become an important model system for studying these processes and for understanding the complex interactions between diet and hormonal pathways.
DOI: http://dx.doi.org/10.7554/eLife.01098.002
doi:10.7554/eLife.01098
PMCID: PMC3810783  PMID: 24175087
mice; aging; caloric restriction; growth hormone; Mouse
6.  Age- and Sex-Associated Plasma Proteomic Changes in Growth Hormone Receptor Gene–Disrupted Mice 
Growth hormone receptor gene–disrupted (GHR−/−) mice are dwarf, insulin sensitive, and long lived despite being obese. In order to identify characteristics associated with their increased longevity, we studied age-related plasma proteomic changes in these mice. Male and female GHR−/− mice and their littermate controls were followed longitudinally at 8, 16, and 24 months of ages for plasma proteomic analysis. Relative to control littermates, GHR−/− mice had increased levels of apolipoprotein A-4 and retinol-binding protein-4 and decreased levels of apolipoprotein E, haptoglobin, and mannose-binding protein-C. Female GHR−/− mice showed decreased inflammatory cytokines including interleukin-1β and monocyte chemotactic protein-1. Additionally, sex differences were found in specific isoforms of apolipoprotein E, RBP-4, haptoglobin, albumin, and hemoglobin subunit beta. In conclusion, we find plasma proteomic changes in GHR−/− mice that favor a longer life span as well as sex differences indicative of an improved health span in female mice.
doi:10.1093/gerona/glr212
PMCID: PMC3403865  PMID: 22156438
Growth hormone receptor; Plasma; Proteomics; Sex; Aging
7.  Bone homeostasis in growth hormone receptor–null mice is restored by IGF-I but independent of Stat5 
Journal of Clinical Investigation  2000;106(9):1095-1103.
Growth hormone (GH) regulates both bone growth and remodeling, but it is unclear whether these actions are mediated directly by the GH receptor (GHR) and/or IGF-I signaling. The actions of GH are transduced by the Jak/Stat signaling pathway via Stat5, which is thought to regulate IGF-I expression. To determine the respective roles of GHR and IGF-I in bone growth and remodeling, we examined bones of wild-type, GHR knockout (GHR–/–), Stat5ab–/–, and GHR–/– mice treated with IGF-I. Reduced bone growth in GHR–/– mice, due to a premature reduction in chondrocyte proliferation and cortical bone growth, was detected after 2 weeks of age. Additionally, although trabecular bone volume was unchanged, bone turnover was significantly reduced in GHR–/– mice, indicating GH involvement in the high bone-turnover level during growth. IGF-I treatment almost completely rescued all effects of the GHR–/– on both bone growth and remodeling, supporting a direct effect of IGF-I on both osteoblasts and chondrocytes. Whereas bone length was reduced in Stat5ab–/– mice, there was no reduction in trabecular bone remodeling or growth-plate width as observed in GHR–/– mice, indicating that the effects of GH in bone may not involve Stat5 activation.
PMCID: PMC301420  PMID: 11067862
8.  Morphology of ovaries in laron dwarf mice, with low circulating plasma levels of insulin-like growth factor-1 (IGF-1), and in bovine GH-transgenic mice, with high circulating plasma levels of IGF-1 
Background
It is well known that somatotrophic/insulin signaling affects lifespan in experimental animals, and one of the signs of aging is progressive gonadal dysfunction.
Methods
To study the effects of insulin-like growth factor-1 (IGF-1) plasma level on ovaries, we analyzed ovaries isolated from 2-year-old growth hormone receptor knockout (GHR-KO) Laron dwarf mice, with low circulating plasma levels of IGF-1, and 6-month-old bovine growth hormone transgenic (bGHTg) mice, with high circulating plasma levels of IGF-1. The ages of the Laron dwarf mutants employed in our studies were selected based on their overall survival (up to ~ 4 years for Laron dwarf mice and ~ 1 year for bGHTg mice).
Results
Morphological analysis of the ovaries of mice that reached ~50% of their maximal life span revealed a lower biological age for the ovaries isolated from 2-year-old Laron dwarf mice than their normal-lifespan wild type littermates. By contrast, the ovarian morphology of increased in size 6 month old bGHTg mice was generally normal.
Conclusion
Ovaries isolated from 2-year-old Laron dwarf mice exhibit a lower biological age compared with ovaries from normal WT littermates at the same age. At the same time, no morphological features of accelerated aging were found in 0.5-year-old bGHTg mice compared with ovaries from normal the same age-matched WT littermates.
doi:10.1186/1757-2215-5-18
PMCID: PMC3583234  PMID: 22747742
Murine ovary; Laron dwarf mouse; Bovine growth hormone transgenic mouse; Growth hormone; Insulin-like growth factor-1; Aging
9.  Age-Related and Depot-Specific Changes in White Adipose Tissue of Growth Hormone Receptor-Null Mice 
Growth hormone receptor-null (GHR−/−) mice are dwarf, insulin sensitive, and long-lived in spite of increased adiposity. However, their adiposity is not uniform, with select white adipose tissue (WAT) depots enlarged. To study WAT depot–specific effects on insulin sensitivity and life span, we analyzed individual WAT depots of 12- and 24-month-old GHR− /− and wild-type (WT) mice, as well as their plasma levels of selected hormones. Adipocyte sizes and plasma insulin, leptin, and adiponectin levels decreased with age in both GHR− /− and WT mice. Two-dimensional gel electrophoresis proteomes of WAT depots were similar among groups, but several proteins involved in endocytosis and/or cytoskeletal organization (Ehd2, S100A10, actin), anticoagulation (S100A10, annexin A5), and age-related conditions (alpha2-macroglobulin, apolipoprotein A-I, transthyretin) showed significant differences between genotypes. Because Ehd2 may regulate endocytosis of Glut4, we measured Glut4 levels in the WAT depots of GHR− /− and WT mice. Inguinal WAT of 12-month-old GHR− /− mice displayed lower levels of Glut4 than WT. Overall, the protein changes detected in this study offer new insights into possible mechanisms contributing to enhanced insulin sensitivity and extended life span in GHR− /− mice.
doi:10.1093/gerona/glt110
PMCID: PMC3859361  PMID: 23873966
Aging; Growth hormone receptor; Adipose tissue depots; Endocytosis; Glut4.
10.  Specific suppression of insulin sensitivity in growth hormone receptor gene-disrupted (GHR-KO) mice attenuates phenotypic features of slow aging 
Aging Cell  2014;13(6):981-1000.
In addition to their extended lifespans, slow-aging growth hormone receptor/binding protein gene-disrupted (knockout) (GHR-KO) mice are hypoinsulinemic and highly sensitive to the action of insulin. It has been proposed that this insulin sensitivity is important for their longevity and increased healthspan. We tested whether this insulin sensitivity of the GHR-KO mouse is necessary for its retarded aging by abrogating that sensitivity with a transgenic alteration that improves development and secretory function of pancreatic β-cells by expressing Igf-1 under the rat insulin promoter 1 (RIP::IGF-1). The RIP::IGF-1 transgene increased circulating insulin content in GHR-KO mice, and thusly fully normalized their insulin sensitivity, without affecting the proliferation of any non-β-cell cell types. Multiple (nonsurvivorship) longevity-associated physiological and endocrinological characteristics of these mice (namely beneficial blood glucose regulatory control, altered metabolism, and preservation of memory capabilities) were partially or completely normalized, thus supporting the causal role of insulin sensitivity for the decelerated senescence of GHR-KO mice. We conclude that a delayed onset and/or decreased pace of aging can be hormonally regulated.
doi:10.1111/acel.12262
PMCID: PMC4326932  PMID: 25244225
endocrinology and metabolism; growth hormone hormonal signaling; insulin sensitivity; longevity regulation; (neuro)endocrinology of senescence
11.  The effect of low and high plasma levels of insulin-like growth factor-1 (IGF-1) on the morphology of major organs: studies of Laron dwarf and bovine growth hormone transgenic (bGHTg) mice 
Histology and histopathology  2013;28(10):1325-1336.
Summary
It is well known that somatotrophic/insulin signaling affects lifespan in experimental animals. To study the effects of insulin-like growth factor-1 (IGF-1) plasma level on the morphology of major organs, we analyzed lung, heart, liver, kidney, bone marrow, and spleen isolated from 2-year-old growth hormone receptor knockout (GHR-KO) Laron dwarf mice (with low circulating plasma levels of IGF-1) and 6-month-old bovine growth hormone transgenic (bGHTg) mice (with high circulating plasma levels of IGF-1). The ages of the two mutant strains employed in our studies were selected based on their overall ~50% survival (Laron dwarf mice live up to ~4 years and bGHTg mice up to ~1 year). Morphological analysis of the organs of long-living 2-year-old Laron dwarf mice revealed a lower biological age for their organs compared with normal littermates, with more brown adipose tissue (BAT) surrounding the main body organs, lower levels of steatosis in liver, and a lower incidence of leukocyte infiltration in different organs. By contrast, the organs of 6-month-old, short-living bGHTg mice displayed several abnormalities in liver and kidney and a reduced content of BAT around vital organs.
PMCID: PMC3988530  PMID: 23613169
Aging; Brown adipose tissue; Insulin-like growth factor-1 (IGF-1); Growth hormone (GH); Laron dwarf mice
12.  Fibroblast Growth Factor 21 Is Not Required for the Reductions in Circulating Insulin-Like Growth Factor-1 or Global Cell Proliferation Rates in Response to Moderate Calorie Restriction in Adult Mice 
PLoS ONE  2014;9(11):e111418.
Calorie restriction (CR) delays aging and extends lifespan in numerous organisms, including mice. Down-regulation of the somatotropic axis, including a reduction in insulin-like growth factor-1 (IGF-1), likely plays an important role in CR-induced lifespan extension, possibly by reducing cell proliferation rates, thereby delaying replicative senescence and inhibiting tumor promotion. Accordingly, elucidating the mechanism(s) by which IGF-1 is reduced in response to CR holds therapeutic potential in the fight against age-related diseases. Up-regulation of fibroblast growth factor 21 (FGF21) is one possible mechanism given that FGF21 expression is induced in response to nutritional deprivation and has been implicated as a negative regulator of IGF-1 expression. Here we investigated alterations in hepatic growth hormone (GH)-mediated IGF-1 production and signaling as well as the role of FGF21 in the regulation of IGF-1 levels and cell proliferation rates in response to moderate CR in adult mice. We found that in response to moderate CR, circulating GH and hepatic janus kinase 2 (JAK2) phosphorylation levels are unchanged but that hepatic signal transducer and activator of transcription 5 (STAT5) phosphorylation levels are reduced, identifying STAT5 phosphorylation as a potential key site of CR action within the somatotropic axis. Circadian measurements revealed that the relative level of FGF21 expression is both higher and lower in CR vs. ad libitum (AL)-fed mice, depending on the time of measurement. Employing FGF21-knockout mice, we determined that FGF21 is not required for the reduction in IGF-1 levels or cell proliferation rates in response to moderate CR. However, compared to AL-fed WT mice, AL-fed FGF21-knockout mice exhibited higher basal rates of cell proliferation, suggesting anti-mitotic effects of FGF21. This work provides insights into both GH-mediated IGF-1 production in the context of CR and the complex network that regulates FGF21 and IGF-1 expression and cell proliferation rates in response to nutritional status.
doi:10.1371/journal.pone.0111418
PMCID: PMC4219748  PMID: 25369265
13.  Growth Hormone Receptor Deficiency is Associated With a Major Reduction in Pro-aging Signaling, Cancer and Diabetes in Humans 
Science Translational Medicine  2011;3(70):70ra13.
Life span extending mutations in growth signaling pathways protect against age-dependent DNA damage in yeast and decrease insulin resistance and cancer in mice. To test their effect in humans, we monitored for 22 years Ecuadorian subjects with mutations in the growth hormone receptor gene leading to severe growth hormone receptor (GHR) and IGF-I deficiencies and combined this information with surveys to identify the cause and age of death for subjects who died before this period. The individuals with GHR deficiency (GHRD) exhibited only one non-lethal malignancy and no cases of diabetes, in contrast to 17% cancer and 5% diabetes prevalence in the controls. A possible explanation for the very low incidence of cancer may be revealed by in vitro studies: serum from GHRD subjects reduced DNA breaks but increased apoptosis in human mammary epithelial cells (HMECs) treated with hydrogen peroxide. We also observed reduced insulin concentrations (1.4 μU/ml vs. 4.4μU/ml in unaffected relatives) and a very low homoeostasis model assessment of insulin resistance (HOMA-IR) index (0.34 vs. 0.96 in unaffected relatives) in GHRD individuals, indicating increased insulin sensitivity, which could explain the absence of diabetes in these subjects. Incubation of HMECs with GHRD serum also resulted in reduced expression of RAS, PKA and TOR, and up-regulation of SOD2, changes that promote cellular protection and life span extension in model organisms. These results provide evidence for a role of evolutionarily conserved pathways in promoting aging and diseases in humans and identify a candidate drug target for healthy life span extension.
doi:10.1126/scitranslmed.3001845
PMCID: PMC3357623  PMID: 21325617
14.  Decreased thyroid follicle size in dwarf mice may suggest the role of growth hormone signaling in thyroid growth regulation 
Thyroid Research  2012;5:7.
Background
Altered somatotrophic signaling is among the most important potential mechanisms of extended longevity. Ames dwarf (df/df) mice are homozygous for mutation at the Prop-1 gene, leading to a lack of growth hormone (GH), prolactin and thyroid stimulating hormone (TSH). Mice homozygous for targeted disruption of the growth hormone receptor/growth hormone binding protein gene are known as GH receptor knockout (GHRKO) mice or “Laron dwarf”. Both, df/df and GHRKO mice, are characterized by reduced body size, low plasma insulin and insulin-like growth factor-I (IGF-I), remarkably extended longevity, and severe (in df/df mice) or mild (in GHRKO mice) thyroid hypofunction. Recently, by crossing df/df and GHRKO mice, double-mutant Ames dwarf/GHRKO (df/KO) mice were created. Interestingly, these mice are smaller than Ames dwarfs or GHRKOs, and also have reduced insulin and IGF-I levels. The aim of the study was to investigate if and to what extent certain thyroid morphological parameters, such as inner follicular surface area, inner follicular perimeter, as well as the follicular epithelium thickness are changed in the examined dwarf mice.
Methods
This quantification was performed in thyroids collected from df/df, GHRKO and df/KO female mice, at approximately 5–6 months of age. We used a computerized plotting programme that combines a live microscopic image of the slide with an operator-generated overlay.
Results
Inner follicular surface area and inner follicular perimeter were decreased in all examined kinds of dwarf mice as compared to normal animals. Furthermore, decreases in these two parameters were more pronounced in df/df and df/KO than in GHRKO mice. Concerning the follicular epithelium thickness, only a tendency towards decrease of this parameter was found in all three kinds of dwarf mice.
Conclusions
Parameters characterizing thyroid follicle size are decreased in all three examined models of dwarf mice, which may explain decreased thyroid hormone levels in both basal mutants (Ames dwarfs and GHRKOs). df/df mutation seems to predominate over GHRKO genetic intervention concerning their effects on thyroid growth. Beside TSH, also GH signaling seems to constitute a crucial element in the regulation of thyroid growth and, possibly, function.
doi:10.1186/1756-6614-5-7
PMCID: PMC3464137  PMID: 22897932
Ames dwarf mice; GHRKO mice; Thyroid follicle; Inner follicular surface area; Inner follicular perimeter; Follicular epithelium thickness
15.  Brain IGF-1 Receptors Control Mammalian Growth and Lifespan through a Neuroendocrine Mechanism 
PLoS Biology  2008;6(10):e254.
Mutations that decrease insulin-like growth factor (IGF) and growth hormone signaling limit body size and prolong lifespan in mice. In vertebrates, these somatotropic hormones are controlled by the neuroendocrine brain. Hormone-like regulations discovered in nematodes and flies suggest that IGF signals in the nervous system can determine lifespan, but it is unknown whether this applies to higher organisms. Using conditional mutagenesis in the mouse, we show that brain IGF receptors (IGF-1R) efficiently regulate somatotropic development. Partial inactivation of IGF-1R in the embryonic brain selectively inhibited GH and IGF-I pathways after birth. This caused growth retardation, smaller adult size, and metabolic alterations, and led to delayed mortality and longer mean lifespan. Thus, early changes in neuroendocrine development can durably modify the life trajectory in mammals. The underlying mechanism appears to be an adaptive plasticity of somatotropic functions allowing individuals to decelerate growth and preserve resources, and thereby improve fitness in challenging environments. Our results also suggest that tonic somatotropic signaling entails the risk of shortened lifespan.
Author Summary
Using a mouse model relevant for humans, we showed that lifespan can be significantly extended by reducing the signaling selectively of a protein called IGF-I in the central nervous system. This effect occurred through changes in specific neuroendocrine pathways. Dissecting the pathophysiological mechanism, we discovered that IGF receptors in the mammalian brain efficiently steered the development of the somatotropic axis, which in turn affected the individual growth trajectory and lifespan. Our work confirms experimentally that continuously low IGF-I and low growth hormone levels favor extended lifespan and postpone age-related mortality. Together with other recent reports, our results further challenge the view that administration of GH can prevent, or even counteract human aging. This knowledge is important since growth hormone is often prescribed to elderly people in an attempt to compensate the unwanted effects of aging. Growth hormone and IGF-I are also substances frequently used for doping in sports.
Inactivating IGF receptors in the brain decreased growth hormone and IGF-I, and increased lifespan in healthy mice. Such neuroendocrine longevity could be a physiological response to environment.
doi:10.1371/journal.pbio.0060254
PMCID: PMC2573928  PMID: 18959478
16.  Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor 
States of growth hormone (GH) resistance, such those observed in Laron’s dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout, GHRKO) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKOHIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1 allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and from poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron’s syndrome does not achieve full skeletal growth.
doi:10.1002/jbmr.1920
PMCID: PMC3843230  PMID: 23456957
IGF-1; growth hormone receptor; bone; micro-computed tomography; betaislet; glucose tolerance
17.  Long-living growth hormone receptor knock out mice: Potential mechanisms of altered stress resistance 
Experimental gerontology  2008;44(1-2):10-19.
Endocrine mutant mice have proven invaluable toward the quest to uncover mechanisms underlying longevity. Growth hormone (GH) and insulin like-growth factor (IGF) have been shown to be key players in physiological systems that contribute to aging processes including glucose metabolism, body composition and cellular protection. Examination of these mutant mice across several laboratories has revealed that differences exist in both the direction and magnitude of change, differences that may result in variation in life span. Growth hormone receptor knock out mice lack a functional GH receptor, therefore GH signaling is absent. These mice have been shown to lack the heightened oxidative defense mechanisms observed in other GH mutants yet live significantly longer than wild type mice. In this study, glutathione (GSH) and methionine (MET) metabolism was examined to determine the extent of variation in this mutant in comparison to the Ames dwarf, a mouse that exhibits delayed aging and life span extension of nearly 70%. Components of GSH and MET were altered in GHR KO compared to wild type controls. The results of these experiments suggest that these pathways may be partially responsible for differences observed in stress resistance and the capacity to respond to stressors, that in the long term, affect health and life span.
doi:10.1016/j.exger.2008.07.002
PMCID: PMC2743895  PMID: 18675334
aging; metabolism; glutathione; methionine; stress resistance
18.  TNF-α downregulates murine hepatic growth hormone receptor expression by inhibiting Sp1 and Sp3 binding 
Journal of Clinical Investigation  2001;107(11):1451-1458.
Children with chronic inflammatory diseases experience growth failure and wasting. This may be due to growth hormone resistance caused by cytokine-induced suppression of growth hormone receptor (GHR) gene expression. However, the factors governing inflammatory regulation of GHR are not known. We have reported that Sp1 and Sp3 regulate hepatic GHR expression. We hypothesized that TNF-α suppresses GHR expression by inhibiting Sp1/Sp3 transactivators. LPS administration significantly reduced murine hepatic GHR expression, as well as Sp1 and Sp3 binding to GHR promoter cis elements. TNF-α was integral to this response, as LPS did not affect hepatic Sp1/Sp3 binding or GHR expression in TNF receptor 1–deficient mice. TNF-α treatment of BNL CL.2 mouse liver cells reduced Sp1 and Sp3 binding to a GHR promoter cis element and downregulated activity of a GHR promoter-driven luciferase reporter. Combined mutations within adjacent Sp elements eliminated GHR promoter suppression by TNF-α without affecting overall nuclear levels of Sp1 or Sp3 proteins. These studies demonstrate that murine GHR transcription is downregulated by LPS, primarily via TNF-α–dependent signaling. Evidence suggests that inhibition of Sp transactivator binding is involved. Further investigation of these mechanisms may identify novel strategies for preventing inflammatory suppression of growth.
PMCID: PMC209317  PMID: 11390427
19.  Neuroendocrine inhibition of glucose production and resistance to cancer in dwarf mice 
Experimental gerontology  2008;44(1-2):26-33.
Pit1 null (Snell dwarf) and Proph1 null (Ames dwarf) mutant mice lack GH, PRL and TSH. Snell and Ames dwarf mice also exhibit reduced IGF-I, resistance to cancer and a longer lifespan than control mice. Endogenous glucose production during fasting is reduced in Snell dwarf mice compared to fasting control mice. In view of cancer cell dependence on glucose for energy, low endogenous glucose production may provide Snell dwarf mice with resistance to cancer. We investigated whether endogenous glucose production is lower in Snell dwarf mice during feeding. Inhibition of endogenous glucose production by glucose injection was enhanced in 12 to 14 month-old female Snell dwarf mice. Thus, we hypothesize that lower endogenous glucose production during feeding and fasting reduces cancer cell glucose utilization providing Snell dwarf mice with resistance to cancer. The elevation of circulating adiponectin, a hormone produced by adipose tissue, may contribute to the suppression of endogenous glucose production in 12 to 14 month-old Snell dwarf mice. We compared the incidence of cancer at time of death between old Snell dwarf and control mice. Only 18% of old Snell dwarf mice had malignant lesions at the time of death compared to 82% of control mice. The median ages at death for old Snell dwarf and control mice were 33 and 26 months, respectively. By contrast, previous studies showed a high incidence of cancer in old Ames dwarf mice at the time of death. Hence, resistance to cancer in old Snell dwarf mice may be mediated by neuroendocrine factors that reduce glucose utilization besides elevated adiponectin, reduced IGF-I and a lack of GH, PRL and TSH, seen in both Snell and Ames dwarf mice. Proteomics analysis of pituitary secretions from Snell dwarf mice confirmed the absence of GH and PRL, the secretion of ACTH and elevated secretion of Chromogranin B and Secretogranin II. Radioimmune assays confirmed that circulating Chromogranin B and Secretogranin II were elevated in 12 to 14 month-old Snell dwarf mice. In summary, our results in Snell dwarf mice suggest that the pituitary gland and adipose tissue are part of a neuroendocrine loop that lowers the risk of cancer during aging by reducing the availability of glucose.
doi:10.1016/j.exger.2008.05.014
PMCID: PMC2872123  PMID: 18582556
Glucose; Cancer; Adiponectin; Growth hormone; Dwarf mice; Pituitary; Chromogranins
20.  Longevity effect of IGF-1R+/− mutation depends on genetic background-specific receptor activation 
Aging Cell  2013;13(1):19-28.
Growth hormone (GH) and insulin-like growth factor (IGF) signaling regulates lifespan in mice. The modulating effects of genetic background gained much attention because it was shown that life-prolonging effects in Snell dwarf and GH receptor knockout vary between mouse strains. We previously reported that heterozygous IGF-1R inactivation (IGF-1R+/−) extends lifespan in female mice on 129/SvPas background, but it remained unclear whether this mutation produces a similar effect in other genetic backgrounds and which molecules possibly modify this effect. Here, we measured the life-prolonging effect of IGF-1R+/− mutation in C57BL/6J background and investigated the role of insulin/IGF signaling molecules in strain-dependent differences. We found significant lifespan extension in female IGF-1R+/− mutants on C57BL/6J background, but the effect was smaller than in 129/SvPas, suggesting strain-specific penetrance of longevity phenotypes. Comparing GH/IGF pathways between wild-type 129/SvPas and C57BL/6J mice, we found that circulating IGF-I and activation of IGF-1R, IRS-1, and IRS-2 were markedly elevated in 129/SvPas, while activation of IGF pathways was constitutively low in spontaneously long-lived C57BL/6J mice. Importantly, we demonstrated that loss of one IGF-1R allele diminished the level of activated IGF-1R and IRS more profoundly and triggered stronger endocrine feedback in 129/SvPas background than in C57BL/6J. We also revealed that acute oxidative stress entails robust IGF-1R pathway activation, which could account for the fact that IGF-1R+/− stress resistance phenotypes are fully penetrant in both backgrounds. Together, these results provide a possible explanation why IGF-1R+/− was less efficient in extending lifespan in C57BL/6J compared with 129/SvPas.
doi:10.1111/acel.12145
PMCID: PMC4326867  PMID: 23898955
Genetic background; gene knockout; IGF-I; IRS; lifespan; stress resistance
21.  Gene expression profiling of long-lived dwarf mice: longevity-associated genes and relationships with diet, gender and aging 
BMC Genomics  2007;8:353.
Background
Long-lived strains of dwarf mice carry mutations that suppress growth hormone (GH) and insulin-like growth factor I (IGF-I) signaling. The downstream effects of these endocrine abnormalities, however, are not well understood and it is unclear how these processes interact with aging mechanisms. This study presents a comparative analysis of microarray experiments that have measured hepatic gene expression levels in long-lived strains carrying one of four mutations (Prop1df/df, Pit1dw/dw, Ghrhrlit/lit, GHR-KO) and describes how the effects of these mutations relate to one another at the transcriptional level. Points of overlap with the effects of calorie restriction (CR), CR mimetic compounds, low fat diets, gender dimorphism and aging were also examined.
Results
All dwarf mutations had larger and more consistent effects on IGF-I expression than dietary treatments. In comparison to dwarf mutations, however, the transcriptional effects of CR (and some CR mimetics) overlapped more strongly with those of aging. Surprisingly, the Ghrhrlit/lit mutation had much larger effects on gene expression than the GHR-KO mutation, even though both mutations affect the same endocrine pathway. Several genes potentially regulated or co-regulated with the IGF-I transcript in liver tissue were identified, including a DNA repair gene (Snm1) that is upregulated in proportion to IGF-I inhibition. A total of 13 genes exhibiting parallel differential expression patterns among all four strains of long-lived dwarf mice were identified, in addition to 30 genes with matching differential expression patterns in multiple long-lived dwarf strains and under CR.
Conclusion
Comparative analysis of microarray datasets can identify patterns and consistencies not discernable from any one dataset individually. This study implements new analytical approaches to provide a detailed comparison among the effects of life-extending mutations, dietary treatments, gender and aging. This comparison provides insight into a broad range of issues relevant to the study of mammalian aging. In this context, 43 longevity-associated genes are identified and individual genes with the highest level of support among all microarray experiments are highlighted. These results provide promising targets for future experimental investigation as well as potential clues for understanding the functional basis of lifespan extension in mammalian systems.
doi:10.1186/1471-2164-8-353
PMCID: PMC2094713  PMID: 17915019
22.  Impact of Food Restriction and Cocaine on Locomotion in Ghrelin- and Ghrelin-Receptor Knockout Mice 
Addiction Biology  2010;16(3):386-392.
Food restriction (FR) augments the behavioral and reinforcing effects of psychomotor stimulants such as cocaine or amphetamine; effects that may be related to the capacity of FR to increase plasma levels of ghrelin (GHR), a 28-amino acid orexigenenic peptide linked to activation of brain dopamine systems. The present study used wild-type (WT) mice or mutant mice sustaining knockout of either GHR (GHR(-/-)) or of the growth hormone secretagogue receptor (GHS-R(-/-)) and subjected to FR or not to evaluate the role of GHR and GHS-R in cocaine-stimulated locomotion. WT, GHR(-/-), and GHS-R(-/-) mice were either restricted to 60% of baseline caloric intake or allowed to free-feed (FF). Mice were treated with 0, 1.25, 2.5 and 5.0 mg/kg cocaine on separate test days (in random dose order) and forward locomotion was recorded on each drug day for 45 min after drug dosing. Food (and water) was available immediately after (but not during) each activity test. For FF mice, there was no interaction between cocaine and GHR status on locomotion. FR-WT mice treated with saline exhibited significant increases in anticipatory locomotion (relative to FF-WT mice), whereas FR-GHS-R(-/-) mice did not. Cocaine significantly increased locomotion in FR-GHR(-/-) and FR-GHS-R(-/-) mice to the levels noted in FR-WT mice. These results suggest that GHS-R activity, but not GHR activity, is required for FR to augment food-associated anticipatory locomotion, but do not support the contention that GHR pathways are required for the capacity of FR to augment the acute effect of cocaine on locomotion.
doi:10.1111/j.1369-1600.2010.00253.x
PMCID: PMC3354738  PMID: 21054685
Food intake; Ghrelin; Psychostimulants
23.  Endocrine Regulation of Heat Shock Protein mRNA Levels in Long-lived Dwarf Mice 
Heat shock proteins (HSPs) maintain proteostasis and may protect against age-associated pathology caused by protein malfolding. In C. elegans, the lifespan extension and thermotolerance in mutants with impaired insulin/IGF signals depends partly on HSP elevation. Less is known about the role of HSPs in the increased lifespan of mice with defects in GH/IGF-I pathways. We measured HSP mRNAs in liver, kidney, heart, lung, muscle and cerebral cortex from long-lived Pit1(dw/dw) Snell dwarf mice. We found many significant differences in HSP mRNA levels between dwarf and control mice, but these effects were complex and organ-specific. We noted 15 instances where HSP mRNAs were lower in Pit1(dw/dw) liver, kidney, or heart tissues, and 14/15 of these were also seen in Ghr(-/-) mice, which lack GH receptor. In contrast, of 12 examples where HSP mRNAs were higher in Snell liver, kidney, or heart, none were altered in Ghr(-/-) mice. Four liver mRNAs were depressed in both Pit1(dw/dw) and Ghr(-/-) mice, and each of these was elevated by GH injection in Ames (Prop1(df/df)) dwarf mice, consistent with the hypothesis that these declines depended on GH and/or IGF-I. Contributions of chaperones to longevity in mice may be more complex than those inferred from C. elegans.
doi:10.1016/j.mad.2009.03.004
PMCID: PMC2718793  PMID: 19428459
24.  Lipopolysaccharide (LPS) directly suppresses growth hormone receptor (GHR) expression through MyD88-dependent and -independent Toll-like receptor-4/MD2 complex signaling pathways 
Introduction
Sepsis is associated with growth hormone (GH) insensitivity and in the intact animal the major surface component of the bacterial cell wall, lipopolysaccharide (LPS), inhibits GH receptor (GHR) gene expression. The prevailing explanation for LPS-induced effects on the GHR promoter is that this effect is indirect via generation of cytokines. Our recent studies demonstrate that saturated free fatty acids (FFAs) inhibit the activity of the murine GHR promoter. Saturated FFAs are an essential component of the lipid A moiety of LPS required for biological activity of LPS.
Hypothesis
LPS directly modulates the activity of the dominant GHR promoter via interaction with Toll-like receptor(s) (TLR)/MD2 complex and activation of cognate signaling pathway(s).
Results
In transient transfection experiments with RAW 264.7 cells which express endogenous TLR4 and MD2, LPS treatment inhibited GHR promoter activity. Co-transfection of dominant negative TLR4 abrogated this effect on GHR promoter activity. In HEK 293T cells, which are devoid of endogenous TLR4 or MD2, ectopic expression of TLR4 and MD2 resulted in LPS-induced inhibition of the GHR promoter activity. The inhibition of GHR promoter activity was demonstrable by 5-6 h after exposure to LPS and persisted at 24 h. Fatty-acid free LPS failed to elicit a similar effect on the GHR promoter and the effect of LPS was abrogated by Polymyxin B. The essential role of the cofactor MD2 on the effect of LPS on the GHR promoter was established in experiments using ectopic expression of wild type and mutant MD2. Cotransfection of CD14 in these cells failed to alter the effect of LPS on the activity of the GHR promoter. Analysis of cell culture supernatant excluded the possibility that the effect of LPS was secondary to release of cytokines from the transfected cells. The effect of LPS on the endogenous GHR promoter activity and protein expression was confirmed in F442A preadipocyte cells. In HEK 293T cells, ectopic expression of mutant MyD88 or mutant TRIF abrogated the effect of LPS on the GHR promoter, suggesting that the effect of LPS on the GHR promoter was via both MyD88-dependent and -independent pathways.
Conclusions
LPS acts through both MyD88-dependent and -independent TLR4 signaling pathways to directly inhibit GHR gene expression. Our results establish a novel cytokine-independent mechanism for decrease in GHR expression in bacterial sepsis.
doi:10.1016/j.mce.2007.05.013
PMCID: PMC1994148  PMID: 17601656
25.  Identification and characterization of single nucleotide polymorphisms in 12 chicken growth-correlated genes by denaturing high performance liquid chromatography 
The genes that are part of the somatotropic axis play a crucial role in the regulation of growth and development of chickens. The identification of genetic polymorphisms in these genes will enable the scientist to evaluate the biological relevance of such polymorphisms and to gain a better understanding of quantitative traits like growth. In the present study, 75 pairs of primers were designed and four chicken breeds, significantly differing in growth and reproduction characteristics, were used to identify single nucleotide polymorphisms (SNP) using the denaturing high performance liquid chromatography (DHPLC) technology. A total of 283 SNP were discovered in 31 897 base pairs (bp) from 12 genes of the growth hormone (GH), growth hormone receptor (GHR), ghrelin, growth hormone secretagogue receptor (GHSR), insulin-like growth factor I and II (IGF-I and -II), insulin-like growth factor binding protein 2 (IGFBP-2), insulin, leptin receptor (LEPR), pituitary-specific transcription factor-1 (PIT-1), somatostatin (SS), thyroid-stimulating hormone beta subunit (TSH-β). The observed average distances in bp between the SNP in the 5'UTR, coding regions (non- and synonymous), introns and 3'UTR were 172, 151 (473 and 222), 89 and 141 respectively. Fifteen non-synonymous SNP altered the translated precursors or mature proteins of GH, GHR, ghrelin, IGFBP-2, PIT-1 and SS. Fifteen indels of no less than 2 bps and 2 poly (A) polymorphisms were also observed in 9 genes. Fifty-nine PCR-RFLP markers were found in 11 genes. The SNP discovered in this study provided suitable markers for association studies of candidate genes for growth related traits in chickens.
doi:10.1186/1297-9686-37-4-339
PMCID: PMC2697233  PMID: 15823239
chickens; genes; SNP; DHPLC

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