Tissue-specific regulation of growth hormone (GH) receptor and insulin-like growth factor-I gene expression in the pituitary and liver of GH-deficient (lit/lit) mice and transgenic mice that overexpress bovine GH (bGH) or a bGH antagonist

GH has diverse biological actions that are mediated by binding to a specific, high-affinity cell surface receptor (GHR). Expression of GHR is tissue specific and a requirement for cellular responsiveness to GH. IGF-I is produced in multiple tissues and regulated in part by GH through GHR. In this study, we evaluated GHR and IGF-I mRNA expression in pituitary gland and compared the levels with those derived from liver of bovine GH transgenic, GH antagonist transgenic, lit/lit mice, and their respective controls using real-time RT-PCR. In liver, both GHR and IGF-I mRNA expressions were regulated in parallel with GH action in all three animal models, and there was a strong correlation between GHR and IGF-I mRNA levels. In the pituitary gland, increased expression of IGF-I mRNA in the pituitary of bovine GH transgenic mice was observed, whereas IGF-I expression in GH antagonist transgenic or lit/lit mice was similar to that observed in control animals. There were no differences of GHR mRNA levels in pituitary gland of any groups we examined. There was also no correlation between GHR and IGF-I mRNA levels in any group in the pituitary gland. In conclusion, we found that hepatic GHR and IGF-I mRNA levels were strongly correlated with each other in chronic GH excess or deficient state, and that regulation and correlation between local GHR and IGF-I mRNA levels induced by GH is different between liver and pituitary gland.     

IL-6-overexpression brings about growth impairment potentially through a GH receptor defect.

This paper is concerned with growth retardation associated with overproduction of interleukin-6 (IL-6). As a model, we used MUP/hIL-6 transgenic mice in which human IL-6 cDNA is overexpressed under the control of a MUP gene enhancer/promoter. The growth-retardation of MUP/hIL-6 transgenic mice was paralleled by reduced serum levels of IGF-I. As shown, hepatic IGF-I mRNA levels were reduced in the transgenic mice. MUP/hIL-6 transgenic mice are in a state of growth hormone (GH)-resistance, since their serum GH levels are either normal or elevated. To identify possible steps in GH signaling which might be perturbed in the transgenic mice, we examined the synthesis of GH receptor (GHR) mRNA. We noted a twofold reduction of hepatic GHR mRNA in the transgenic mice. We therefore conclude that overexpression of IL-6 brings about growth impairment in part through a GH receptor defect.     

Thyroid hormones are involved in insulin-like growth factor-I (IGF-I) production by stimulating hepatic growth hormone receptor (GHR) gene expression in the chicken.

Effect of thyroid status on IGF-I production in growing chickens was studied. Serum concentrations of GH were not affected by propylthiouracil (PTU) or thyroxine (T4) treatments, whereas serum IGF-I levels were significantly decreased in PTU-treated chickens. The lowered serum IGF-I levels in the PTU-treated group were completely restored to the control levels by T4 injections. In the liver, the messenger RNA (mRNA) expressions both for GH receptor (GHR) and IGF-I were significantly repressed by PTU treatment, and were restored again by T4 replacement. In addition, the results of analysis on radiolabelled GH binding to the liver membrane were consistent with the levels of hepatic GHR mRNA expression. Serum concentrations of IGF-I were positively correlated with hepatic IGF-I mRNA and GHR mRNA expressions. The correlation coefficient between serum T3 levels and hepatic IGF-I mRNA expressions was also significant. These results indicate that thyroid hormones regulate IGF-I production in the chicken by affecting hepatic GHR expression.     

Diurnal variation in growth hormone receptor messenger ribonucleic acid in liver and skeletal muscle of lit/+ and lit/lit mice

The present study investigated the diurnal variation in GH receptor (GHR) mRNA in liver and skeletal muscle of 3-month-old GH-deficient and -sufficient mice using quantitative real-time RT-PCR. lit/lit (GH deficient) or lit/+ (GH sufficient) mice were fed ad libitum and lights were on between 0600 and 2000. Tissues were collected at 0800-1000, 1200-1400 and 2000-2200. Hepatic GHR mRNA levels of lit/+ mice at 0800-1000 were significantly lower than those at 1200-1400 and 2000-2200. There was no significant variation in hepatic GHR mRNA of lit/lit mice. In skeletal muscle, GHR mRNA levels of both lit/+ and lit/lit mice at 1200-1400 were significantly higher than those at 0800-1000 and 2000-2200. There was also a diurnal change in hepatic IGF-I mRNA levels of lit/+ but not of lit/lit mice; the levels were lowest at 0800-1000 in lit/+ mice. On the other hand, there was no variation in IGF-I mRNA levels in skeletal muscle. These results suggest that 1) there is a diurnal variation in GHR expression in liver and skeletal muscle and the pattern of the variation is tissue specific; 2) GH deficiency blunted the diurnal variation in GHR mRNA in liver but not that in skeletal muscle; 3) IGF-I mRNA expression in liver is more closely related to GHR mRNA expression than that in skeletal muscle.     

The decrease in hepatic IGF-I gene expression in arthritic rats is not associated with modifications in hepatic GH receptor mRNA

OBJECTIVE: Adjuvant-induced arthritis induces a catabolic response, and a decrease in circulating IGF-I. Hypermetabolism and GH insensitivity have been described in acute inflammation. The aim of this study was to analyze whether impaired IGF-I secretion in arthritic rats can be attributed to hepatic GH resistance. DESIGN AND METHODS: Male Wistar rats were injected with complete Freund's adjuvant, and 14 days afterwards arthritic and control rats were injected daily with recombinant human GH (rhGH) (3 IU/kg) or saline for 8 days. GH receptor (GHR) gene expression in the liver and the effect of rhGH on hepatic IGF-I synthesis in arthritic rats were examined. RESULTS: There was a significant decrease in hepatic concentrations of IGF-I (P < 0.01) as well as in the IGF-I gene expression in arthritic but not in pair-fed rats. In contrast, arthritis did not modify GHR mRNA levels in the liver. The 8 day administration of rhGH resulted in an increase in body weight gain in arthritic but not in control rats. There was an increase in hepatic IGF-I synthesis and in GHR mRNA levels after rhGH treatment, both in control and in arthritic rats. Two endotoxin lipopolysaccharide (LPS) (1 mg/kg) injections decreased hepatic concentrations of IGF-I and IGF-I mRNA (P < 0.01). Contrary to the results obtained in arthritic rats, mRNA expression of GHR in the liver was lower in LPS- than in saline-treated rats (P < 0.01). CONCLUSION: These data suggest that the decrease in IGF-I synthesis induced by chronic arthritis is not secondary to GH resistance.     

The GH/IGF-I axis in the brushtail possum (Trichosurus vulpecula) pouch young

Plasma and pituitary GH concentrations and liver GH receptor (GHR), IGF-I and IGF-binding protein-3 (IGFBP-3) mRNA expression were determined in brushtail possum (Trichosurus vulpecula) pouch young aged 12-150 days post-partum and in adults. Mean plasma GH concentrations were highest, measuring around 150 ng/ml, from 12 to 100 days post-partum, and thereafter declined so that by 150 days post-partum levels were not significantly different from those in adults (10.8+/-1.8 ng/ml (S.E.M.)). In contrast to plasma levels, pituitary GH content increased markedly throughout pouch life, with an 87-fold increase between 12 and 150 days post-partum. However, when expressed per gram body weight, pituitary content was relatively constant between 25 and 150 days post-partum, indicating that the decline in plasma GH after 100 days post-partum was not due to decreased synthesis and/or storage of GH in the pituitary gland. Expression of GHR, IGF-I and IGFBP-3 mRNAs was determined by semi-quantitative RT-PCR. Liver GHR and IGF-I mRNA expression were low at 12 and 25 days post-partum and did not show sustained and significant increases (P<0.05) until 125 and 150 days post-partum. IGFBP-3 expression was also low at 12 days post-partum but then increased rapidly to a maximum at 50 days post-partum and thereafter declined. For all three mRNAs, liver expression at day 150 was not significantly different from that in adults. These patterns of gene expression for GHR and IGF-I suggest that the possum liver is resistant to the high plasma GH concentrations during early pouch life and in this way is similar to the fetal liver of some eutherian mammals.     

Effects of fasting on growth hormone, growth hormone receptor, and insulin-like growth factor-I axis in seawater-acclimated tilapia, Oreochromis mossambicus

Effects of fasting on the growth hormone (GH)--growth hormone receptor (GHR)-insulin-like growth factor-I (IGF-I) axis were characterized in seawater-acclimated tilapia (Oreochromis mossambicus). Fasting for 4 weeks resulted in significant reductions in body weight and specific growth rate. Plasma GH and pituitary GH mRNA levels were significantly elevated in fasted fish, whereas significant reductions were observed in plasma IGF-I and hepatic IGF-I mRNA levels. There was a significant negative correlation between plasma levels of GH and IGF-I in the fasted fish. No effect of fasting was observed on hepatic GHR mRNA levels. Plasma glucose levels were reduced significantly in fasted fish. The fact that fasting elicited increases in GH and decreases in IGF-I production without affecting GHR expression indicates a possible development of GH resistance.     

Dexamethasone inhibits both growth hormone (GH)-induction of insulin-like growth factor-I (IGF-I) mRNA and GH receptor (GHR) mRNA levels in rat primary cultured hepatocytes.

Glucocorticoids are potent inhibitors of growth. In this work, we investigated whether glucocorticoids inhibit the stimulatory action of GH on IGF-I gene expression in rat hepatocytes. GH increased IGF-I mRNA levels 11-fold after 24 h, whereas high doses of DXM (10(-6)M) caused a slight (2.6-fold) increase of IGF-I mRNA levels. However, high doses of DXM (10(-6)M) inhibited the induction of IGF-I mRNA by GH. To assess the role of GHR in this inhibition, we investigated the regulation of GHR expression. High doses of DXM decreased GHR mRNA levels. This effect was already detectable 6 h after addition of 10(-6)M DXM and was dose-dependent, with a maximal inhibition observed at a concentration of 10(-6)M. In conclusion, our results show that high doses of DXM inhibits the GH-induced IGF-I gene expression and the GHR gene expression. The parallel decrease of GHR and GH-induced IGF-I mRNA suggests that the GH resistance caused by DXM is mediated by diminished GH receptor synthesis.     

Dexamethasone inhibits growth hormone induction of insulin-like growth factor-I (IGF-I) messenger ribonucleic acid (mRNA) in hypophysectomized rats and reduces IGF-I mRNA abundance in the intact rat

Exogenous glucocorticoids are potent inhibitors of skeletal growth in experimental animals and children. The mechanism whereby glucocorticoids exert this effect is unclear, and circulating somatomedin levels have been reported to be low, normal, or high in this situation. Since recent studies have emphasized the importance of autocrine or paracrine insulin-like growth factor I (IGF-I), we have examined the effects of dexamethasone (DXM) on IGF-I gene expression in the hypophysectomized (hypox) and pituitary intact rat. An increase in IGF-I messenger RNA (mRNA) abundance of approximately 5-, 3-, 2-, and 1.5-fold in the liver, proximal tibia, lung, and kidney, respectively, was seen in hypox rats killed 6 h after injection of human GH (100 micrograms/100 g body wt). As little as 1 micrograms/100 g body wt of DXM administered 3 h before GH injection significantly reduced GH induction of IGF-I mRNA in the liver and the proximal tibia (P less than 0.05 and P less than 0.005, respectively). Higher doses of DXM were required to reduce IGF-I mRNA abundance in the kidney and lung. Of the tissues examined the order of sensitivity to DXM was liver greater than tibia greater than kidney greater than lung. In contrast to the effects of DXM on tissue IGF-I mRNA abundance, an approximately 10-fold higher dose of DXM was required to inhibit the GH-induced rise in serum IGF-I concentration. The effect of DXM on steady state IGF-I mRNA abundance in pituitary-intact rats which were killed 9 h after DXM was also examined. The reduction in IGF-I mRNA abundance required higher doses of DXM (6-360 micrograms/100 g body wt) and was less marked in pituitary-intact rats than in GH-treated hypox rats. In the pituitary-intact rats the order of sensitivity to DXM was tibia greater than liver greater than lung greater than kidney. In acute studies serum IGF-I levels were not decreased by any dose of DXM; rather a significant increase in serum IGF-I was apparent in pituitary intact rats, treated with the lowest and highest doses of DXM. When DXM was administered daily for 6 days to pituitary intact rats, body wt gain and hepatic and tibial IGF-I mRNA abundance were significantly reduced. No significant changes were seen in serum IGF-I concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Liver-derived IGF-I regulates GH secretion at the pituitary level in mice

We have reported that liver-specific deletion of IGF-I in mice (LI-IGF-I-/-) results in decreased circulating IGF-I and increased GH levels. In the present study, we determined how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion. The pituitary mRNA levels of GH releasing factor (GHRF) receptor and GH secretagogue (GHS) receptor were increased in LI-IGF-I-/- mice, and in line with this, their GH response to ip injections of GHRF and GHS was increased. Expression of mRNA for pituitary somatostatin receptors, hypothalamic GHRF, somatostatin, and neuropeptide Y was not altered in LI-IGF-I-/- mice, whereas hypothalamic IGF-I expression was increased. Changes in hepatic expression of major urinary protein and the PRL receptor in male LI-IGF-I-/- mice indicated an altered GH release pattern most consistent with enhanced GH trough levels. Liver weight was enhanced in LI-IGF-I-/- mice of both genders. In conclusion, loss of liver-derived IGF-I enhances GH release by increasing expression of pituitary GHRF and GHS receptors. The enhanced GH release in turn affects several liver parameters, in line with the existence of a pituitary-liver axis.     

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.     

Glucocorticoids are not necessary for the inhibitory effect of endotoxic shock on serum IGF-I and hepatic IGF-I mRNA

The aim of this work was to elucidate the possible role of glucocorticoids in the bacterial lipopolysaccharide (LPS)-induced decrease in hepatic IGF-I synthesis. For this purpose, we studied the effect of LPS on IGF-I in two rat strains, Wistar and Lewis, which have different adrenal responses to inflammation. Compared with Wistar rats, Lewis rats have a reduced hypothalamic-pituitary-adrenal response to inflammatory stimuli. Rats received two i.p. injections of 1 mg/kg LPS and were killed 4 h after the second injection. LPS induced an increase in serum concentrations of both ACTH and corticosterone, the increase being more pronounced in Wistar than in Lewis rats. LPS decreased hepatic GH receptor (GHR) and IGF-I mRNA only in Wistar rats. However, serum concentrations of IGF-I were significantly decreased (P<0.01) in both Wistar and Lewis rats. These data indicate that the adrenal axis may mediate the inhibitory effect of LPS on GHR and IGF-I synthesis in the liver. In a second experiment, adrenalectomized or sham-operated Wistar rats were injected with LPS. Two LPS injections (0.1 mg/kg) decreased serum concentrations of IGF-I in both type of rat; however, the inhibitory effect of LPS on liver GHR and IGF-I mRNA was observed in adrenalectomized rats, but not in intact rats. All these data suggest that some component of the adrenal axis, other than glucocorticoids, mediates the inhibitory effect of LPS on liver GHR and IGF-I.     

Developmental expression of hepatic growth hormone receptor and insulin-like growth factor-I mRNA in the chicken.

We have examined the ontogeny of expression of growth hormone (GH) receptor (GHR) and insulin-like growth factor-I (IGF-I) mRNA in chicken liver from day 13 of incubation until 31 weeks of age. The profiles of GHR and IGF-I mRNA levels were compared to developmental changes in body weight and plasma levels of GH and IGF-I. In the embryo, hepatic GHR mRNA was not detectable until day 15, highest on days 17 and 19, and then declined at hatching (day 21). Following an initial 2-week delay after hatching, there was a progressive increase in hepatic GHR mRNA which continued after the birds reached mature body weight. Plasma GH reached peak levels at 3-4 weeks of age and then fell sharply until maintenance of a low basal level after 10 weeks of age. Thus, there appears to be a strong inverse relationship between expression of the GHR and basal plasma GH levels in the prepubertal chicken. Although IGF-I mRNA was undetectable in embryonic liver by Northern blot analysis, there is a good correlation between expression of hepatic IGF-I mRNA and the plasma IGF-I profile during post-hatching development in the chicken. The highest levels of IGF-I mRNA were reached at 4 weeks of age which was followed by a slow decline to the basal levels maintained after 10 weeks of age. It appears that the decline in plasma IGF-I lags considerably behind the sharp fall in plasma GH levels and expression of hepatic IGF-I mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The growth hormone (GH)-axis of GH receptor/binding protein gene-disrupted and metallothionein-human GH-releasing hormone transgenic mice: hypothalamic neuropeptide and pituitary receptor expression in the absence and presence of GH feedback

Elevation of circulating GH acts to feed back at the level of the hypothalamus to decrease GH-releasing hormone (GHRH) and increase somatostatin (SRIF) production. In the rat, GH-induced changes in GHRH and SRIF expression are associated with changes in pituitary GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R), and SRIF receptor subtype messenger RNA (mRNA) levels. These observations suggest that GH regulates its own synthesis and release not only by altering expression of key hypothalamic neuropeptides but also by modulating the sensitivity of the pituitary to hypothalamic input, by regulating pituitary receptor synthesis. To further explore this possibility, we examined the relationship between the expression of hypothalamic neuropeptides [GHRH, SRIF, and neuropeptide Y (NPY)] and pituitary receptors [GHRH-R, GHS-R, and SRIF receptor subtypes (sst2 and sst5)] in two mouse strains with alterations in the GH-axis; the GH receptor/binding protein gene-disrupted mouse (GHR/BP-/-) and the metallothionein promoter driven human GHRH (MT-hGHRH) transgenic mouse. In GHR/BP-/- mice, serum insulin-like growth factor I levels are low, and circulating GH is elevated because of the lack of GH negative feedback. Hypothalamic GHRH mRNA levels in GHR/BP-/- mice were 232 +/- 20% of GHR/BP+/+ littermates (P < 0.01), whereas SRIF and NPY mRNA levels were reduced to 86 +/- 2% and 52 +/- 3% of controls, respectively (P < 0.05; ribonuclease protection assay). Pituitary GHRH-R and GHS-R mRNA levels of GHR/BP-/- mice were elevated to 275 +/- 55% and 319 +/- 68% of GHR/BP+/+ values (P < 0.05, respectively), whereas the sst2 and sst5 mRNA levels did not differ from GHR/BP intact controls as determined by multiplex RT-PCR. Therefore, in the absence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor stimulation of GH synthesis and release. In MT-hGHRH mice, ectopic hGHRH transgene expression elevates circulating GH and insulin-like growth factor I. In this model of GH excess, endogenous (mouse) hypothalamic GHRH mRNA levels were reduced to 69 +/- 6% of nontransgenic controls, whereas SRIF mRNA levels were increased to 128 +/- 6% (P < 0.01). NPY mRNA levels were not significantly affected by hGHRH transgene expression. Also, MT-hGHRH pituitary GHRH-R and GHS-R mRNA levels did not differ from controls. However, sst2 and sst5 mRNA levels in MT-hGHRH mice were increased to 147 +/- 18% and 143 +/- 16% of normal values, respectively (P < 0.05). Therefore, in the presence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor suppression of GH synthesis and release.