Regulation of skeletal growth and mineral acquisition by the GH/IGF-1 axis: Lessons from mouse models.

The growth hormone (GH) and its downstream mediator, the insulin-like growth factor-1 (IGF-1), construct a pleotropic axis affecting growth, metabolism, and organ function. Serum levels of GH/IGF-1 rise during pubertal growth and associate with peak bone acquisition, while during aging their levels decline and associate with bone loss. The GH/IGF-1 axis was extensively studied in numerous biological systems including rodent models and cell cultures. Both hormones act in an endocrine and autocrine/paracrine fashion and understanding their distinct and overlapping contributions to skeletal acquisition is still a matter of debate. GH and IGF-1 exert their effects on osteogenic cells via binding to their cognate receptor, leading to activation of an array of genes that mediate cellular differentiation and function. Both hormones interact with other skeletal regulators, such as sex-steroids, thyroid hormone, and parathyroid hormone, to facilitate skeletal growth and metabolism. In this review we summarized several rodent models of the GH/IGF-1 axis and described key experiments that shed new light on the regulation of skeletal growth by the GH/IGF-1 axis.

Liver-derived IGF-I regulates mean life span in mice.

BACKGROUND: Transgenic mice with low levels of global insulin-like growth factor-I (IGF-I) throughout their life span, including pre- and postnatal development, have increased longevity. This study investigated whether specific deficiency of liver-derived, endocrine IGF-I is of importance for life span. METHODS AND FINDINGS: Serum IGF-I was reduced by approximately 80% in mice with adult, liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice), and body weight decreased due to reduced body fat. The mean life span of LI-IGF-I(-/-) mice (n = 84) increased 10% vs. control mice (n = 137) (Cox's test, p<0.01), mainly due to increased life span (16%) of female mice [LI-IGF-I(-/-) mice (n = 31): 26.7±1.1 vs. control (n = 67): 23.0±0.7 months, p<0.001]. Male LI-IGF-I(-/-) mice showed only a tendency for increased longevity (p = 0.10). Energy expenditure, measured as oxygen consumption during and after submaximal exercise, was increased in the LI-IGF-I(-/-) mice. Moreover, microarray and RT-PCR analyses showed consistent regulation of three genes (heat shock protein 1A and 1B and connective tissue growth factor) in several body organs in the LI-IGF-I(-/-) mice. CONCLUSIONS: Adult inactivation of liver-derived, endocrine IGF-I resulted in moderately increased mean life span. Body weight and body fat decreased in LI-IGF-I(-/-) mice, possibly due to increased energy expenditure during exercise. Genes earlier reported to modulate stress response and collagen aging showed consistent regulation, providing mechanisms that could underlie the increased mean life span in the LI-IGF-I(-/-) mice.

The role of liver-derived insulin-like growth factor-I.

IGF-I is expressed in virtually every tissue of the body, but with much higher expression in the liver than in any other tissue. Studies using mice with liver-specific IGF-I knockout have demonstrated that liver-derived IGF-I, constituting a major part of circulating IGF-I, is an important endocrine factor involved in a variety of physiological and pathological processes. Detailed studies comparing the impact of liver-derived IGF-I and local bone-derived IGF-I demonstrate that both sources of IGF-I can stimulate longitudinal bone growth. We propose here that liver-derived circulating IGF-I and local bone-derived IGF-I to some extent have overlapping growth-promoting effects and might have the capacity to replace each other (= redundancy) in the maintenance of normal longitudinal bone growth. Importantly, and in contrast to the regulation of longitudinal bone growth, locally derived IGF-I cannot replace (= lack of redundancy) liver-derived IGF-I for the regulation of a large number of other parameters including GH secretion, cortical bone mass, kidney size, prostate size, peripheral vascular resistance, spatial memory, sodium retention, insulin sensitivity, liver size, sexually dimorphic liver functions, and progression of some tumors. It is clear that a major role of liver-derived IGF-I is to regulate GH secretion and that some, but not all, of the phenotypes in the liver-specific IGF-I knockout mice are indirect, mediated via the elevated GH levels. All of the described multiple endocrine effects of liver-derived IGF-I should be considered in the development of possible novel treatment strategies aimed at increasing or reducing endocrine IGF-I activity.

Liver-derived IGF1 enhances the androgenic response in prostate.

Both IGF1 and androgens are major enhancers of prostate growth and are implicated in the development of prostate hyperplasia and cancer. The aim of the present study was to investigate whether liver-derived endocrine IGF1 modulates the androgenic response in prostate. Mice with adult, liver-specific inactivation of IGF1 (LI-IGF1(-/-) mice) displayed an approximately 80% reduction in serum IGF1 levels associated with decreased prostate weight compared with control mice (anterior prostate lobe -19%, P<0.05; dorsolateral prostate (DLP) lobe -35%, P<0.01; ventral prostate (VP) lobe -47%, P<0.01). Reduced androgen receptor (Ar) mRNA and protein levels were observed in the VP lobe (-34% and -30% respectively, both P<0.05 versus control mice). Analysis of prostate morphology showed reductions in both the glandular and fibromuscular compartments of the VP and DLP lobes that were proportional to the reductions in the weights of these lobes. Immunohistochemistry revealed reduced intracellular AR immunoreactivity in the VP and DLP lobes. The non-aromatizable androgen dihydrotestosterone increased VP weight to a lesser extent in orchidectomized (ORX) LI-IGF1(-/-) mice than in ORX controls (-40%, P<0.05 versus control mice). In conclusion, deficiency of liver-derived IGF1 reduces both the glandular and fibromuscular compartments of the prostate, decreases AR expression in prostate, and reduces the stimulatory effect of androgens on VP weight. These findings may explain, at least in part, the well-known clinical association between serum IGF1 levels and conditions with abnormal prostate growth.

Liver-derived IGF-I regulates kidney size, sodium reabsorption, and renal IGF-II expression.

The GH/-IGF-I axis is important for kidney size and function and may also be involved in the development of renal failure. In this study, the role of liver-derived endocrine IGF-I for kidney size and function was investigated in mice with adult liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice). These mice have an 80-85% reduction of serum IGF-I level and compensatory increased GH secretion. Seven-month-old as well as 24-month-old LI-IGF-I(-/-) mice had decreased kidney weight. Glomerular filtration rate, assessed using creatinine clearance as well as creatinine clearance corrected for body weight, was unchanged. The 24-h urine excretion of sodium and potassium was increased in the LI-IGF-I(-/-) mice. In the 24-month-old mice, there was no between-group difference in kidney morphology. Microarray and real-time PCR (RT-PCR) analyses showed a high renal expression of IGF-II in the control mice, whereas in the LI-IGF-I(-/-) mice, there was a tissue-specific decrease in the renal IGF-II mRNA levels (-79%, P < 0.001 vs controls using RT-PCR). In conclusion, deficiency of circulating liver-derived IGF-I in mice results, despite an increase in GH secretion, in a global symmetrical decrease in kidney size, increased urinary sodium and potassium excretion, and a clear down regulation of renal IGF-II expression. However, the LI-IGF-I(-/-) mice did not develop kidney failure or nephrosclerosis. One may speculate that liver-derived endocrine IGF-I induces renal IGF-II expression, resulting in symmetrical renal growth.

Growth hormone overexpression in the central nervous system results in hyperphagia-induced obesity associated with insulin resistance and dyslipidemia.

It is well known that peripherally administered growth hormone (GH) results in decreased body fat mass. However, GH-deficient patients increase their food intake when substituted with GH, suggesting that GH also has an appetite stimulating effect. Transgenic mice with an overexpression of bovine GH in the central nervous system (CNS) were created to investigate the role of GH in CNS. This study shows that overexpression of GH in the CNS differentiates the effect of GH on body fat mass from that on appetite. The transgenic mice were not GH-deficient but were obese and showed increased food intake as well as increased hypothalamic expression of agouti-related protein and neuropeptide Y. GH also had an acute effect on food intake following intracerebroventricular injection of C57BL/6 mice. The transgenic mice were severely hyperinsulinemic and showed a marked hyperplasia of the islets of Langerhans. In addition, the transgenic mice displayed alterations in serum lipid and lipoprotein levels and hepatic gene expression. In conclusion, GH overexpression in the CNS results in hyperphagia-induced obesity indicating a dual effect of GH with a central stimulation of appetite and a peripheral lipolytic effect.

GH, IGF-I, and growth.

Recent studies have confirmed the importance of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis as the major determinant of whole body growth in animals and humans and have highlighted the significance of IGF-I to human growth. Pharmacological studies in rodents and therapeutic studies in humans demonstrate that recombinant human (rh)IGF-I can induce a significant statural growth response. Mouse gene knockout studies have shown that IGF-I, rather than GH, is the major hormone controlling whole body growth. The relative importance of endocrine versus local IGF-I remains unresolved. In children who are GH- and IGF-I-deficient, treatment with GH stimulates a robust growth response. In children who are IGF-I-deficient but GH-sufficient, rhIGF-I might also be a useful treatment. Furthermore, pharmacological and genetic studies in animals show effects requiring the combined presence of GH and IGF-I, suggesting that rhGH and rhIGF-I together might be the optimal treatment for some children with short stature.

Development of pure prolactin receptor antagonists.

Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all forms of PRL receptor antagonists obtained to date exhibit partial agonism, preventing their therapeutic use as full antagonists. In the present study, we describe the development of new human PRL antagonists devoid of agonistic properties and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including highly sensitive assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as assessed by analyzing their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland, and prostate). Finally, by using transgenic mice expressing PRL specifically in the prostate, which exhibit constitutively activated signaling cascades paralleling hyperplasia, we show that these new PRL analogs are able to completely revert PRL-activated events. These second generation human PRL antagonists are good candidates to be used as inhibitors of growth-promoting actions of PRL.

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.

Expression of delta opioid receptor mRNA and protein in the rat cerebral cortex and cerebellum is decreased by growth hormone.

Hormones released from the pituitary have been shown to regulate the expression of different proteins in the central nervous system. We wanted to examine whether peripheral administration of bovine growth hormone (bGH) regulates the expression of delta-opioid receptor (DOR) in the cerebral cortex and cerebellum. Expression of the DOR protein was quantified using Western blot densitometry. DOR mRNA was quantified with a solution hybridization RNase protection assay. Hypophysectomized (Hx) and untreated normal female rats were included in the study. All Hx rats were hormonally treated with cortisol (400 microg/kg/day) and L-thyroxine (10 microg/kg/day) for 19 days. Hypophysectomy resulted in a threefold increase in cerebral cortex and a twofold increase in cerebellum of the DOR protein compared with normal rats. One subgroup of Hx rats received bGH (1 mg/kg body weight) as a daily subcutaneous injection for 19 days. This treatment normalized the levels of DOR protein in the cerebral cortex and cerebellum. Immunohistochemical experiments showed that GH decreased DOR expression especially in layers II-VI in cerebral cortex and in stratum moleculare in cerebellum. Quantification of DOR mRNA by solution hybridization RNase protection assay corresponded to the DOR protein measurements. We conclude that the expression of DORs in cerebral cortex and cerebellum is regulated by GH.

The growth hormone-binding protein is a location-dependent cytokine receptor transcriptional enhancer.

In the rat, a growth hormone-binding protein (GHBP) exists that is derived from the growth hormone (GH) receptor gene by an alternative mRNA splicing mechanism such that the transmembrane and intracellular domains of the GH receptor are replaced by a hydrophilic carboxyl terminus. In isolation, the GHBP is inactive, although it does compete with the receptor for ligand binding in the extracellular space and therefore inhibits the cellular response to GH. The GHBP is also located intracellularly and is translocated to the nucleus upon ligand stimulation. We show here that endogenously produced GHBP, in contrast to exogenous GHBP, was able to enhance the STAT5-mediated transcriptional response to GH. Interestingly, when the GHBP was targeted constitutively to the nucleus by the addition of the nuclear localization sequence of the SV40 large T antigen, greater enhancement of STAT5-mediated transcription was obtained. The transcriptional enhancement did not require GH per se and was not specific to the GH receptor, since similar enhancement of STAT5-mediated transcription by nuclear localized GHBP was obtained with specific ligand stimulation of both prolactin and erythropoietin receptors. Thus, the GHBP exerts divergent effects on STAT5-mediated transcription depending on its cellular location. The use of a soluble cytokine receptor as a location-dependent transcriptional enhancer and the ligand-independent involvement of the extracellular domain of a polypeptide ligand receptor in intracellular signal transduction provide additional novel mechanisms of transcriptional control.

Effects of liver-derived insulin-like growth factor I on bone metabolism in mice.

Insulin-like growth factor (IGF) I is an important regulator of both skeletal growth and adult bone metabolism. To better understand the relative importance of systemic IGF-I versus locally expressed IGF-I we have developed a transgenic mouse model with inducible specific IGF-I gene inactivation in the liver (LI-IGF-I-/-). These mice are growing normally up to 12 weeks of age but have a disturbed carbohydrate and lipid metabolism. In this study, the long-term effects of liver-specific IGF-I inactivation on skeletal growth and adult bone metabolism were investigated. The adult (week 8-55) axial skeletal growth was decreased by 24% in the LI-IGF-I-/- mice whereas no major reduction of the adult appendicular skeletal growth was seen. The cortical cross-sectional bone area, as measured in the middiaphyseal region of the long bones, was decreased in old LI-IGF-I-/- mice. This reduction in the amount of cortical bone was caused mainly by decreased periosteal circumference and was associated with a weaker bone determined by a decrease in ultimate load. In contrast, the amount of trabecular bone was not decreased in the LI-IGF-I-/- mice. DNA microarray analysis of 30-week-old LI-IGF-I-/- and control mice indicated that only four genes were regulated in bone whereas approximately 40 genes were regulated in the liver, supporting the hypothesis that liver-derived IGF-I is of minor importance for adult bone metabolism. In summary, liver-derived IGF-I exerts a small but significant effect on cortical periosteal bone growth and on adult axial skeletal growth while it is not required for the maintenance of the trabecular bone in adult mice.

Liver-derived insulin-like growth factor-I is involved in the regulation of blood pressure in mice.

IGF-I has been suggested to be of importance for cardiovascular structure and function, but the relative role of locally produced and liver-derived endocrine IGF-I remains unclear. Using the Cre-LoxP recombination system, we have previously created transgenic mice with a liver-specific, inducible IGF-I knockout (LI-IGF-I-/-). To examine the role of liver-derived IGF-I in cardiovascular physiology, liver-derived IGF-I was inactivated at 4 wk of age, resulting in a 79% reduction of serum IGF-I levels. At 4 months of age, systolic blood pressure (BP) was increased in LI-IGF-I-/- mice. Echocardiography showed increased posterior wall thickness in combination with decreased stroke volume and cardiac output, whereas other systolic variables were unchanged, suggesting that these cardiac effects were secondary to increased peripheral resistance. Acute nitric oxide-synthase inhibition increased systolic BP more in LI-IGF-I-/- mice than in control mice. LI-IGF-I-/- mice showed impaired acetylcholine-induced vasorelaxation in mesenteric resistance vessels and increased levels of endothelin-1 mRNA in aorta. Thus, the increased peripheral resistance in LI-IGF-I-/- mice might be attributable to endothelial dysfunction associated with increased expression of endothelin-1 and impaired vasorelaxation of resistance vessels. In conclusion, our findings suggest that liver-derived IGF-I is involved in the regulation of BP in mice.

A model for tissue-specific inducible insulin-like growth factor-I (IGF-I) inactivation to determine the physiological role of liver-derived IGF-I.

Insulin-like growth factor-I (IGF-I) has important growthpromoting and metabolic effects and is expressed in virtually every tissue of the body. The highest expression is found in the liver, but the physiological role of liver-derived IGF-I is unknown. It has been difficult to separate the endocrine effects of liver-derived IGF-I from the autocrine/paracrine effects of locally produced IGF-I in peripheral tissues. Therefore, we have developed a mouse model with a liver-specific inducible deletion of the IGF-I gene (LI-IGF-I-/- mouse). The LI-IGF-I-/- mouse has dramatically reduced (>80%) serum IGF-I levels, demonstrating that the major part of serum IGF-I is liver-derived. Surprisingly, LI-IGFI -/- mice demonstrate a normal appendicular skeletal growth up to at least 12 mo of age despite the dramatic decrease in circulating IGF-I levels, indicating that liver-derived IGF-I is not required for appendicular skeletal growth. However, the adult axial skeletal growth is reduced in the LI-IGF-I-/- mice. Furthermore, the amount of cortical bone is reduced due to decreased radial growth of the cortical bone, while the trabecular bone mineral density is unchanged in the LI-IGFI -/- mice. The decreased levels of circulating IGF-I are associated with increased serum levels of growth hormone (GH), indicating a role for liver-derived IGFI in the negative-feedback regulation of GH secretion. Measurements of factors regulating GH secretion in the pituitary and in the hypothalamus revealed an increased expression of GH-releasing-hormone (GHRH) and GHsecretagogue (GHS) receptors in the pituitary of LI-IGFI -/- mice. This in turn results in an increased sensitivity to systemically administered GHRH and GHS, demonstrating that the regulatory action of liver-derived IGF-I on GH secretion is at the pituitary rather than at the hypothalamic level. The liver is an important metabolic organ and LI-IGF-I-/- mice are markedly hyperinsulinemic and yet normoglycemic, consistent with an adequately compensated insulin resistance. Interestingly, LI-IGF-I-/- mice display a reduced age-dependent fat mass accumulation compared with control mice. Furthermore, LI-IGF-I-/- mice have increased blood pressure attributable to increased peripheral resistance indicating a role for liver-derived IGF-I in the regulation of blood pressure. In conclusion, liver-derived IGF-I is important for carbohydrate and lipid metabolism and for the regulation of GH secretion at the pituitary level. Furthermore, it regulates adult axial skeletal growth and cortical radial growth while it is not required for appendicular skeletal growth.