Mice Overexpressing Growth Hormone Research Articles

Growth hormone upregulates the pro-tumorigenic galectin 1 in mouse liver.

Transgenic mice overexpressing growth hormone (GH) spontaneously develop liver tumors, including hepatocellular carcinoma (HCC), within a year. The preneoplastic liver pathology in these mice recapitulates that observed in humans at high risk of developing hepatic cancer. Although increased expression of galectin 1 (GAL1) in liver tissue is associated with HCC aggressiveness, a link between this glycan-binding protein and hormone-related tumor development has not yet been explored. In this study, we investigated GAL1 expression during liver tumor progression in mice continuously exposed to high levels of GH. GAL1 expression was determined by Western blotting, RT-qPCR and immunohistochemistry in the liver of transgenic mice overexpressing GH. Animals of representative ages at different stages of liver pathology were studied. GAL1 expression was upregulated in the liver of GH-transgenic mice. This effect was observed at early ages, when animals displayed no signs of liver disease or minimal histopathological alterations and was also detected in young adults with preneoplastic liver pathology. Remarkably, GAL1 upregulation was sustained during aging and its expression was particularly enhanced in liver tumors. GH also induced hepatic GAL1 expression in mice that were treated with this hormone for a short period. Moreover, GH triggered a rapid increment in GAL1 protein expression in human HCC cells, denoting a direct effect of the hormone on hepatocytes. Therefore, our results indicate that GH upregulates GAL1 expression in mouse liver, which may have critical implications in tumorigenesis. These findings suggest that this lectin could be implicated in hormone-driven liver carcinogenesis.

Mice overexpressing growth hormone exhibit increased skeletal muscle myostatin and MuRF1 with attenuation of muscle mass

BackgroundIn contrast to the acute effects of growth hormone (GH) on skeletal muscle protein synthesis, long-term GH treatment appears to have negligible effects on muscle mass. Despite this knowledge, little is known regarding the chronic effects of GH on skeletal muscle protein synthesis and atrophy signaling pathways. The purpose of this study was to determine if protein synthesis pathways are attenuated and/or muscle atrophy intracellular signaling pathways are altered in the skeletal muscle of transgenic bovine GH (bGH) mice.MethodsThe gastrocnemius and soleus from 5-month-old male bGH mice (n = 9) and wild type (WT) controls (n = 9) were harvested and analyzed for proteins involved in the protein synthesis (Akt/mTOR), growth and proliferation (MAPK), and muscle atrophy (MuRF1 and myostatin) pathways.ResultsTotal body mass was significantly increased in bGH mice compared to WT controls (49%, P < 0.0001). When expressed relative to total body mass, the gastrocnemius (− 28%, P < 0.0001), but not the soleus, was significantly lower in mice overexpressing GH, compared to controls. Transgenic bGH mice had elevated phosphorylation levels of protein kinase b (Akt1), 4E-binding protein 1 (4E-BP1), p70 S6 kinase, p42/44, and p38 (P < 0.05) compared to WT littermates. Mature myostatin (26 kDa), premature myostatin (52 kDa), and activin receptor type IIB (AcvR2B) protein levels were increased in bGH mice (P < 0.05), along with elevated phosphorylation levels of mothers against decapentaplegic homolog (Smad2) (59%, P < 0.0001). Mice overexpressing GH had increased MuRF1 expression (30%, P < 0.05) and insulin receptor substrate 1 (IRS1) serine phosphorylation (44%, P < 0.05) in the gastrocnemius, but not the soleus, when compared to controls.ConclusionsThese findings demonstrate that chronic elevations in circulating GH have a critical impact on signaling pathways involved in skeletal muscle protein synthesis and atrophy, and suggest that MuRF1, myostatin, and IRS1 serine phosphorylation may act to inhibit exaggerated glycolytic muscle growth, in environments of chronic GH/IGF-1 excess.

Attenuation of epidermal growth factor (EGF) signaling by growth hormone (GH).

Transgenic mice overexpressing growth hormone (GH) show increased hepatic protein content of the epidermal growth factor receptor (EGFR), which is broadly associated with cell proliferation and oncogenesis. However, chronically elevated levels of GH result in desensitization of STAT-mediated EGF signal and similar response of ERK1/2 and AKT signaling to EGF compared to normal mice. To ascertain the mechanisms involved in GH attenuation of EGF signaling and the consequences on cell cycle promotion, phosphorylation of signaling mediators was studied at different time points after EGF stimulation, and induction of proteins involved in cell cycle progression was assessed in normal and GH-overexpressing transgenic mice. Results from kinetic studies confirmed the absence of STAT3 and 5 activation and comparable levels of ERK1/2 phosphorylation upon EGF stimulation, which was associated with diminished or similar induction of c-MYC, c-FOS, c-JUN, CYCLIN D1 and CYCLIN E in transgenic compared to normal mice. Accordingly, kinetics of EGF-induced c-SRC and EGFR phosphorylation at activating residues demonstrated that activation of these proteins was lower in the transgenic mice with respect to normal animals. In turn, EGFR phosphorylation at serine 1046/1047, which is implicated in the negative regulation of the receptor, was increased in the liver of GH-overexpressing transgenic mice both in basal conditions and upon EGF stimulus. Increased basal phosphorylation and activation of the p38-mitogen-activated protein kinase might account for increased Ser 1046/1047 EGFR. Hyperphosphorylation of EGFR at serine residues would represent a compensatory mechanism triggered by chronically elevated levels of GH to mitigate the proliferative response induced by EGF.

Transgenic Mice Overexpressing Growth Hormone (GH) Have Reduced or Increased Cardiac Apoptosis through Activation of Multiple GH-Dependent or -Independent Cell Death Pathways

GH has antiapoptotic effects in cardiac or noncardiac cell lines; however, increased apoptosis has been found in myocardial samples of patients with acromegaly. The aim of this study was to investigate cardiac apoptosis and underlying molecular mechanisms in transgenic mice overexpressing bovine GH [acromegalic mice (Acro)] aged 3 or 9 months. Cardiomyocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase assay and annexin V; expression of pro- or antiapoptotic proteins was assessed by Western blot. Specificity of GH action was confirmed using a selective GH receptor antagonist. Apoptosis was lower in 3-month-old Acro than in controls; reduction was abolished by a GH receptor antagonist. The effects of GH were consistent with an antiapoptotic phenotype (increased Bcl2 and Bcl-XL and reduced Bad and cytochrome c levels, leading to lower activation of caspase-9 and caspase-3). In contrast, apoptosis was higher in 9-month-old Acro than in littermate controls; in addition, a GH receptor antagonist was without effect; the proapoptotic phenotype consisted in increased Bad, cytochrome c, caspase-9, and caspase-3. GH reduced apoptosis through p38 and p44/42 kinase pathways at young ages, whereas phosphatidylinositol-3-kinase was silent; on the contrary, the effects of GH on p38 and p44/42 kinase pathways were overcome by GH-independent stimuli in 9-month-old Acro. In addition, the antiapoptotic effect of GH was still present at this age as shown by phosphatidylinositol-3-kinase/Akt pathway activation. In conclusion, chronic GH excess reduced apoptosis at a young age, whereas its antiapoptotic action was overwhelmed in older animals by GH-independent mechanisms, leading to increased cell death.

Systemic overexpression of growth hormone (GH) in transgenic FVB/N inbred mice: an optimized model for holistic studies of molecular mechanisms underlying GH-induced kidney pathology

Transgenic mice overexpressing growth hormone (GH) display a plethora of phenotypic alterations and provide unique models for studying and influencing consequences of chronic GH excess. Since the first report on GH transgenic mice was published in 1982, many different mouse models overexpressing GH from various species at different levels and with different tissue specificities were established, most of them on random-bred or hybrid genetic background. We have generated a new transgenic mouse model on FVB/N inbred background, expressing bovine (b) GH under the control of the chicken beta-actin promoter (cbetaa). cbetaa-bGH transgenic mice exhibit ubiquitous expression of bGH mRNA and protein and circulating bGH levels in the range of several microg/ml, resulting in markedly stimulated growth and the characteristic spectrum of pathological lesions which were described in previous GH overexpressing mouse models. Importantly, a consistent sequence of renal alterations is observed, mimicking progressive kidney disease in human patients. The novel, genetically standardized GH transgenic mouse model is ideal for holistic transcriptome and proteome studies aiming at the identification of the molecular mechanisms underlying GH-induced pathological alterations especially in the kidney. Moreover, genetically defined cbetaa-bGH mice facilitate random mutagenesis screens for modifier genes which influence the effects of chronic GH excess and associated pathological lesions.

A dietary supplement abolishes age-related cognitive decline in transgenic mice expressing elevated free radical processes.

We previously found that transgenic mice overexpressing growth hormone (TGM) have elevated and progressively increasing free radical processes in brain that strongly correlates with reduced survivorship. Young mature TGM, however, displayed vastly enhanced learning of an eight-choice cued maze and qualitatively different learning curves than normal controls. Here we document the age-related patterns in learning ability of TGM and normal mice. Learning appeared inferior in both genotypes of very young mice but TGM were confirmed to be superior to normal mice upon maturity. Older TGM, however, showed rapid age-related loss of their exceptional learning, whereas normal mice at 1 year of age showed little change. The cognitive decline of TGM was abolished by a complex "anti-aging" dietary supplement formulated to promote membrane and mitochondrial integrity, increase insulin sensitivity, reduce reactive oxygen and nitrogen species, and ameliorate inflammation. Results are discussed in the context of reactive oxygen and nitrogen species, long-term potentiation, learning, aging and neuropathology, based on known impacts of the growth hormone axis on the brain, and characteristics of TGM.

Bovine growth hormone-transgenic mice have major alterations in hepatic expression of metabolic genes.

Transgenic mice overexpressing growth hormone (GH) have been extensively used to study the chronic effects of elevated serum levels of GH. GH is known to have many acute effects in the liver, but little is known about the chronic effects of GH overexpression on hepatic gene expression. Therefore, we used DNA microarray to compare gene expression in livers from bovine GH (bGH)-transgenic mice and littermates. Hepatic expression of peroxisome proliferator-activated receptor-alpha (PPARalpha) and genes involved in fatty acid activation, peroxisomal and mitochondrial beta-oxidation, and production of ketone bodies was decreased. In line with this expression profile, bGH-transgenic mice had a reduced ability to form ketone bodies in both the fed and fasted states. Although the bGH mice were hyperinsulinemic, the expression of sterol regulatory element-binding protein (SREBP)-1 and most lipogenic enzymes regulated by SREBP-1 was reduced, indicating that these mice are different from other insulin-resistant models with respect to expression of SREBP-1 and its downstream genes. This study also provides several candidate genes for the well-known association between elevated GH levels and cardiovascular disease, e.g., decreased expression of scavenger receptor class B type I, hepatic lipase, and serum paraoxonase and increased expression of serum amyloid A-3 protein. We conclude that bGH-transgenic mice display marked changes in hepatic genes coding for metabolic enzymes and suggest that GH directly or indirectly regulates many of these hepatic genes via decreased expression of PPARalpha and SREBP-1.

Modulation of transforming growth factor beta1 gene expression in the mammary gland by insulin-like growth factor I and octreotide.

Transforming growth factor beta1 (TGF-beta1) has been shown to exhibit anti-proliferative activity for mammary gland epithelial cells and for human breast cancer cells. Insulin-like growth factor I (IGF-I), in contrast, is a well-characterized mitogenic and anti-apoptotic factor involved in mammary gland physiology. In order to examine the hypothesis that IGF-I suppresses TGF-beta1 expression in the mammary gland, we studied the effect of various manipulations of the growth hormone - IGF-I axis on TGF-beta1 mRNA abundance. Administration of IGF-I to intact animal suppressed TGF-beta1 mRNA levels in a dose-dependent manner to approximately 20% of control levels. Administration of the somatostatin analogue octreotide in a manner previously shown to acutely suppress the growth hormone - IGF-I axis increased mammary gland TGF-beta1 expression approximately 3-fold. Transgenic mice overexpressing growth hormone expressed TGF-beta1 in the mammary gland at only approximately 12% of the level of control animals, while mice IGF-I deficient due to the mutation expressed TGF-beta1 at slightly higher levels than control animals. The large differences in TGF-beta1 expression between control and GH-transgenic animals were correlated with major differences in architecture of the mammary gland, while the appearance of mammary glands of normal and animals was similar. These data document a previously unrecognized relationship between TGF-beta1 and IGF-I physiology in the mammary gland, and suggest a novel mechanism by which somatostatin analogues influence the proliferative behaviour of breast epithelial cells.

Effects of growth hormone overexpression and growth hormone resistance on neuroendocrine and reproductive functions in transgenic and knock-out mice.

Transgenic mice overexpressing growth hormone (GH) exhibit alterations in the function of the hypothalamic-pituitary-gonadal (HPG) axis and the H-P-adrenal axis. Alterations in the turnover of hypothalamic neurotransmitters, in plasma hormone levels, and in regulation of their release are associated with reproductive deficits, particularly in females. Results reported after publication of our minireview on this subject provided evidence that GH-transgenic mice have increased binding of GH to GH binding proteins in plasma, are hyperinsulinemic and insulin resistant, and have major alterations in energy budgets with increased allocation to growth. Reduced life span and fertility of these animals may be related to insufficient allocation of energy to reproduction and maintenance. Growth hormone resistance induced by transgenic expression of an antagonistic bGH analog or by targeted disruption (knock-out, KO) of the GH receptor (GH-R) gene leads to dramatic suppression of plasma levels of insulin-like growth factor-1 (IGF-1), and dwarf phenotype due to reduced growth and increased adiposity. In both models of GH resistance, there are marked reproductive deficits in females, decline of breeding performance of males, and alterations in the function of the HPG axis. In GH-R-KO females, puberty is delayed, and litter size is reduced. Fetal weights are reduced whereas placental weights are increased, and the weight of newborn pups is reduced despite an increase in the length of gestation. In GH-R-KO males, copulatory behavior and fertility are reduced, plasma PRL is elevated, and responses to luteinizing hormone releasing hormone (LHRH) in vivo and to LH in vitro are suppressed. However, reproductive deficits in GH-R-KO mice are very mild when compared to those described previously in IGF-KO animals. Apparently, the amounts of IGF-1 that may be produced locally in the absence of GH stimulation are sufficient for sexual maturation and fertility in both sexes, whereas quantitative deficits in reproductive function reflect absence of GH-dependent IGF-1 production and other consequences of eliminating GH signaling. The reproduction phenotype of the GH-R-KO mice is also mild when compared to dwarf mice that lack GH, prolactin (PRL), and thyroid stimulating hormone (TSH). This is presumably related to the presence of redundant mechanisms in the stimulatory control of the gonads by the pituitary and the ability of animals capable of producing PRL and TSH to compensate partially for the absence of GH signaling.

Linear bone growth of oMT1a-oGH transgenic male mice.

Linear bone growth was studied in male mice possessing a controlled ovine metallothionein 1a promoter-ovine growth hormone (oMT1a-oGH) transgene. Transgene expression was activated at weaning by the addition of 25 mM zinc sulfate to drinking water; transgenic and control mice received the zinc supplementation. The ulna, humerus, and tibia were excised at 10-day intervals until 130 days from control and from mice hemizygous for oMT1a-oGH. Bones from mice overexpressing growth hormone (GH) were 11-20% longer than those from controls (P less than 0.01) at 130 days. Transgenic mice exhibited both an enhanced rate of bone growth and a growth period of greater duration, i.e., the ulna, tibia, and humerus from oMT1a-oGH mice grew at an accelerated rate for an additional 20-40 days relative to the same bones from control mice. The bones from both groups were characterized by isometric growth patterns. Genetic size scaling revealed that the observed differences in bone growth were directly related to the mature size of the bone, suggesting that the bones possess an inherent growth pattern that is followed even in the presence of elevated GH.