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1.  Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis 
Hepatology (Baltimore, Md.)  2015;61(2):613-626.
Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the Growth hormone receptor gene (Ghr-/-, a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2-/-), a mouse model of inflammatory cholestasis and liver fibrosis.
Ghr-/-;Mdr2-/- mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation and increased collagen deposition relative to Mdr2 -/- mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr-/-;Mdr2-/- mice had a pronounced down-regulation of hepato-protective genes Hnf6, Egfr and Igf-1, and significantly increased levels of ROS and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr-/-) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis and bile infarcts compared to their wildtype littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr-/-;Mdr2-/- mice displayed a significant decrease in tumour incidence compared to Mdr2-/- mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver.
Conclusion
Our findings suggest that GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments.
doi:10.1002/hep.27408
PMCID: PMC4986903  PMID: 25179284
ROS; IGF-1; cancer; apoptosis; bile acids
2.  Growth hormone modulates hypothalamic inflammation in long‐lived pituitary dwarf mice 
Aging Cell  2015;14(6):1045-1054.
Summary
Mice in which the genes for growth hormone (GH) or GH receptor (GHR −/−) are disrupted from conception are dwarfs, possess low levels of IGF‐1 and insulin, have low rates of cancer and diabetes, and are extremely long‐lived. Median longevity is also increased in mice with deletion of hypothalamic GH‐releasing hormone (GHRH), which leads to isolated GH deficiency. The remarkable extension of longevity in hypopituitary Ames dwarf mice can be reversed by a 6‐week course of GH injections started at the age of 2 weeks. Here, we demonstrate that mutations that interfere with GH production or response, in the Snell dwarf, Ames dwarf, or GHR −/− mice lead to reduced formation of both orexigenic agouti‐related peptide (AgRP) and anorexigenic proopiomelanocortin (POMC) projections to the main hypothalamic projection areas: the arcuate nucleus (ARH), paraventricular nucleus (PVH), and dorsomedial nucleus (DMH). These mutations also reduce hypothalamic inflammation in 18‐month‐old mice. GH injections, between 2 and 8 weeks of age, reversed both effects in Ames dwarf mice. Disruption of GHR specifically in liver (LiGHRKO), a mutation that reduces circulating IGF‐1 but does not lead to lifespan extension, had no effect on hypothalamic projections or inflammation, suggesting an effect of GH, rather than peripheral IGF‐1, on hypothalamic development. Hypothalamic leptin signaling, as monitored by induction of pStat3, is not impaired by GHR deficiency. Together, these results suggest that early‐life disruption of GH signaling produces long‐term hypothalamic changes that may contribute to the longevity of GH‐deficient and GH‐resistant mice.
doi:10.1111/acel.12382
PMCID: PMC4693470  PMID: 26268661
aging; dwarf mice; growth hormone; hypothalamus; inflammation; longevity
3.  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
4.  Exon 3-deleted and full-length growth hormone receptor polymorphism frequencies in an Iranian population 
The functional role of the exon 3 growth hormone receptor (d3GHR) polymorphism in human and its distributions in different populations is not clearly understood. The presence of full length growth hormone (flGHR) is the most important in metabolic risk factors. The aim of this study was to define the frequency distribution of d3GHR/full-length GHR in an Iranian population. The presence of the d3GHR polymorphism in healthy volunteers blood DNA (n=80, male=30 and female=50) was assessed by PCR using specific primers. The 935-bp and 592-bp fragments indicate the presence of the flGHR and the exon3 deletion of GHR, respectively. The distribution of the GHR genotypes in this study were 31.4% (n=24) for fl/flGHR, 49.7 % (n=41) for fl/d3GHR, and 19.0 % (n=15) for d3/d3GHR. Frequencies of fl allele and d3 allele were 55.4% and 44.4% within whole population, respectively. There was no difference in allels frequencies of GHR in male (fl=0.583, d3=0.417) and female (fl=0.540, d3=0.460) when compared with whole population. The results showed that the frequency of d3/d3GHR isoform was significantly lower than that of the fl/flGHR and d3/flGHR. The frequencies of GHR polymorphisms were likely consistent with previous reports. Our finding is also consistant with Mexican population. The advantage of existence of the d3/d3 rather than fl/flGHR polymorphisms in individuals and in correlation with diseases opens new insights for GH and insilin-like-growth factor-1 (IGF-I) axis.
PMCID: PMC4326986  PMID: 26339263
d3GHR; d3GHR/flGHR; Polymorphism; Population; GH/IGF-I axis
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  Osteoblast-restricted Disruption of the Growth Hormone Receptor in Mice Results in Sexually Dimorphic Skeletal Phenotypes 
Bone Research  2013;1(1):85-97.
Growth hormone (GH) exerts profound anabolic actions during postnatal skeletal development, in part, through stimulating the production of insulin-like growth factor-1 (IGF-1) in liver and skeletal tissues. To examine the requirement for the GH receptor (GHR) in osteoblast function in bone, we used Cre-LoxP methods to disrupt the GHR from osteoblasts, both in vitro and in vivo. Disruption of GHR from primary calvarial osteoblasts in vitro abolished GH-induced signaling, as assessed by JAK2/STAT5 phosphorylation, and abrogated GH-induced proliferative and anti-apoptotic actions. Osteoblasts lacking GHR exhibited reduced IGF-1-induced Erk and Akt phosphorylation and attenuated IGF-1-induced proliferation and anti-apoptotic action. In addition, differentiation was modestly impaired in osteoblasts lacking GHR, as demonstrated by reduced alkaline phosphatase staining and calcium deposition. In order to determine the requirement for the GHR in bone in vivo, we generated mice lacking the GHR specifically in osteoblasts (ΔGHR), which were born at the expected Mendelian frequency, had a normal life span and were of normal size. Three week-old, female ΔGHR mice had significantly reduced osteoblast numbers, consistent with the in vitro data. By six weeks of age however, female ΔGHR mice demonstrated a marked increase in osteoblasts, although mineralization was impaired; a phenotype similar to that observed previously in mice lacking IGF-1R specifically in osteoblasts. The most striking phenotype occurred in male mice however, where disruption of the GHR from osteoblasts resulted in a “feminization” of bone geometry in 16 week-old mice, as observed by μCT. These results demonstrate that the GHR is required for normal postnatal bone development in both sexes. GH appears to serve a primary function in modulating local IGF-1 action. However, the changes in bone geometry observed in male ΔGHR mice suggest that, in addition to facilitating IGF-1 action, GH may function to a greater extent than previously appreciated in establishing the sexual dimorphism of the skeleton.
doi:10.4248/BR201301006
PMCID: PMC4472095  PMID: 26273494
growth hormone; osteoblasts; knockout mice; bone; sexual dimorphism
13.  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.
14.  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
15.  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
16.  Expression of Apoptosis-Related Genes in Liver-Specific Growth Hormone Receptor Gene–Disrupted Mice Is Sex Dependent 
Apoptosis is a process that affects life span and health. Mice with liver-specific disruption of the growth hormone receptor (GHR) gene (ie, Ghr gene) liver-specific growth hormone receptor knockout [LiGHRKO] mice), as opposed to mice with global deletion of the Ghr gene (GHRKO; Ghr−/−), are characterized by severe hepatic steatosis and lack of improved insulin sensitivity. We have previously shown that levels of proapoptotic factors are decreased in long-lived and insulin-sensitive GHRKO mice. In the current study, expression of specific apoptosis-related genes was assessed in brains, kidneys, and livers of male and female LiGHRKO and wild-type mice using real-time PCR. In the brain, expression of Caspase 3, Caspase 9, Smac/DIABLO, and p53 was decreased in females compared with males. Renal expression of Caspase 3 and Noxa also decreased in female mice. In the liver, no differences were seen between males and females. Also, no significant genotype effects were detected in the examined organs. Lack of significant genotype effect in kidneys contrasts with previous observations in GHRKO mice. Apparently, global GHR deletion induces beneficial changes in apoptotic factors, whereas liver-specific GHR disruption does not. Furthermore, sexual dimorphism may play an important role in regulating apoptosis during liver-specific suppression of the somatotrophic signaling.
doi:10.1093/gerona/glu008
PMCID: PMC4296163  PMID: 24550353
Apoptosis; Growth hormone receptor (GHR); GHR gene disruption; Knockout mice.
17.  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
18.  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
19.  Growth Hormone Influence on the Morphology and Size of the Mouse Meibomian Gland 
Journal of Ophthalmology  2016;2016:5728071.
Purpose. We hypothesize that growth hormone (GH) plays a significant role in the regulation of the meibomian gland. To test our hypothesis, we examined the influence of GH on mouse meibomian gland structure. Methods. We studied four groups of mice, including (1) bovine (b) GH transgenic mice with excess GH; (2) GH receptor (R) antagonist (A) transgenic mice with decreased GH; (3) GHR knockout (−/−) mice with no GH activity; and (4) wild type (WT) control mice. After mouse sacrifice, eyelids were processed for morphological and image analyses. Results. Our results show striking structural changes in the GH-deficient animals. Many of the GHR−/− and GHA meibomian glands featured hyperkeratinized and thickened ducts, acini inserting into duct walls, and poorly differentiated acini. In contrast, the morphology of WT and bGH meibomian glands appeared similar. The sizes of meibomian glands of bGH mice were significantly larger and those of GHA and GHR−/− mice were significantly smaller than glands of WT mice. Conclusions. Our findings support our hypothesis that the GH/IGF-1 axis plays a significant role in the control of the meibomian gland. In addition, our data show that GH modulates the morphology and size of this tissue.
doi:10.1155/2016/5728071
PMCID: PMC4769763  PMID: 26981277
20.  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
21.  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
22.  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
23.  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
24.  Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor 
A series of antagonists specifically targeting growth hormone receptors (GHR) in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH). Therefore, in this study, we developed and characterized a porcine GHR (pGHR) antibody antagonist (denoted by AN98) via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1) secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.
doi:10.5713/ajas.15.0892
PMCID: PMC5003978  PMID: 26954133
Porcine Growth Hormone; Porcine Growth Hormone Receptors; Signalling Pathway; Antagonist
25.  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

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