Continuous Growth Hormone Administration Research Articles

Growth hormone and Insulin-like growth factor-I (IGF-I) modulate the expression of L-type amino acid transporters in the muscles of spontaneous dwarf rats and L6 and C2C12 myocytes

ObjectiveBranched-chain amino acids (BCAAs) have been reported to inhibit several types of muscle atrophy via the activation of the mechanistic target of rapamycin complex 1 (mTORC1). However, we previously found that BCAA did not activate mTORC1 in growth hormone (GH)-deficient spontaneous dwarf rats (SDRs), and that GH restored the stimulatory effect of BCAAs toward the mTORC1. The objective of this study was to determine whether GH or Insulin-like growth factor-I (IGF-I) stimulated the expression of L-type amino acid transporters (LATs) that delivered BCAAs, and whether LATs were involved in the mTORC1 activation. DesignAfter the continuous administration of GH, cross-sectional areas (CSAs) of muscle fibers and LAT mRNA levels in the skeletal muscles of SDRs were compared to those from the SDRs that received normal saline. The effect of GH and IGF-I on LAT mRNA levels were determined in L6 and C2C12 myocytes. The effects of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), a blocker for LATs, and LAT1 siRNA on mTORC1 activation and cell functions were examined in C2C12 cells. ResultsGH increased LAT1 and LAT4 mRNA levels in accordance with the increase in CSAs of muscle fibers in SDRs. IGF-I, and not GH, increased LAT1 mRNA levels in cultured L6 myocytes. IGF-I also increased LAT1 mRNA level in another myocyte line, C2C12. Furthermore, IGF-I reduced LAT3 and LAT4 mRNA levels in both cell lines. GH reduced LAT3 and LAT4 mRNA levels in L6 cells. BCH decreased basal C2C12 cell proliferation and reduced IGF-I-induced phosphorylation of 4E-BP1 and S6K, both of which are mTORC1 targets, but LAT1 siRNA did not affect the phosphorylation. This suggests that BCH may exert its effect via other pathway than LAT1.Conclusions: IGF-I increased LAT1 mRNA level in myocytes. However, the role of LAT1 in IGF-I-induced mTORC1 activation and cell functions remains unclear.

Regulation of Human CYP2C18 and CYP2C19 in Transgenic Mice: Influence of Castration, Testosterone, and Growth Hormone

The hormonal regulation of human CYP2C18 and CYP2C19, which are expressed in a male-specific manner in liver and kidney in a mouse transgenic model, was examined. The influence of prepubertal castration in male mice and testosterone treatment of female mice was investigated, as was the effect of continuous administration of growth hormone (GH) to transgenic males. Prepubertal castration of transgenic male mice suppressed the expression of CYP2C18 and CYP2C19 in liver and kidney to female levels, whereas expression was increased for the endogenous female-specific mouse hepatic genes Cyp2c37, Cyp2c38, Cyp2c39, and Cyp2c40. Testosterone treatment of female mice increased CYP2C18 and CYP2C19 expression in kidney, and to a lesser extent in liver, but was without effect in brain or small intestine, where gene expression was not gender-dependent. Continuous GH treatment of transgenic males for 7 days suppressed hepatic expression of CYP2C19 (>90% decrease) and CYP2C18 ( approximately 50% decrease) but had minimal effect on the expression of these genes in kidney, brain, or small intestine. Under these conditions, continuous GH induced all four female-specific mouse liver Cyp2c genes in males to normal female levels. These studies indicate that the human CYP2C18 and CYP2C19 genes contain regulatory elements that respond to the endogenous mouse hormonal profiles, with androgen being the primary regulator of male-specific expression in kidney, whereas the androgen-dependent pituitary GH secretory pattern is the primary regulator of male-specific expression in liver in a manner that is similar to the regulation of the endogenous gender-specific hepatic genes.

Continuous administration of growth hormone does not prevent the decrease of IGF-I gene expression in zinc-deprived rats despite normalization of liver GH binding

To determine the role of reduced liver GH binding (GHR) in the decreased IGF-I observed in zinc-deficient (ZD) animals, we investigated the effects of GHR restoration on growth, insulin-like growth factor I (IGF-I) and its binding proteins (IGFBPs) in ZD rats. Rats were fed for 4 weeks a zinc-deficient diet (ZD Zn, 0 ppm) or a Zinc-normal diet (pair-fed or PF; Zn, 75 ppm). ZD rats received continuous s.c. infusion of bovine growth hormone (bGH) (100 microg/d) for the 4 weeks or for the last week of the study. Compared with pair-fed rats, zinc deficiency produced attenuated weight gain (-43%, P < 0.001), lower serum IGF-I and liver IGF-I mRNA (-52%, P < 0.001 and -44%, P < 0.05), lower serum IGFBPs (IGFBP-3 -66%, IGFBP-4 -48%, 34-29 kDa IGFBP cluster -53%, P < 0.05), lower liver GHR and its mRNA (-20 and -34%, P < 0.05) and lower serum growth hormone binding protein (GHBP) and its mRNA (-56 and -48%, P < 0.05; all comparisons vs PF rats). Exogenous bGH given continuously normalized the liver GHR, serum GHBP and their liver mRNAs, as well as circulating IGFBPs. Despite restoration of GHR and GHBP to normal, growth, serum IGF-I and its liver mRNA were not stimulated by GH infusion in ZD rats, indicating that IGF-I synthesis requires the presence of zinc in addition to GH, and that the lack of growth-promoting action of GH in zinc-deprived rats results from a defect beyond GH binding to its liver receptors.

Chronic administration of growth hormone (GH) to adult chickens exerts marked effects on circulating concentrations of insulin-like growth factor-I (IGF-I), IGF binding proteins, hepatic GH regulated gene I, and hepatic GH receptor mRNA.

In young birds, growth hormone (GH) administration has been found to have only a small or even no effect on circulating concentrations of insulin-like growth factor-I (IGF-I). This is in obvious contrast to the situation in mammals. The present study examines the effect of continuous administration of GH in adult male chickens. Plasma concentrations of IGF-I were markedly elevated (2.5-3.0-fold, p < 0.001) in GH-treated chickens. There were also some transient increases in the circulating levels of IGF binding proteins. Adult chickens showed other manifestations of increased responsiveness to GH, including elevated hepatic expression of GH-regulated gene-I (mRNA) with GH treatment (p < 0.05), and a tendency (p < 0.08) for decreased GH-receptor mRNA. In contrast to the changes in circulating concentrations of GH and IGF-I with GH treatment, no changes in plasma concentrations of thyroid hormones, reproductive hormones, glucose, or nonesterified fatty acids were evident.

Zonation of cytochrome P450 enzyme expression in rat liver: Isozyme-specific regulation by pituitary dependent hormones

The effect of hypophysectomy and subsequent infusion of growth hormone (GH) or injections of triiodothyronine (T 3) on the acinar expression pattern of four hormonally regulated P450 isozymes was studied to elucidate the involvement of pituitary dependent hormones in regulating the characteristic centrilobular expression pattern of most members of the cytochrome P450 ( CYP) gene family in rat liver. Hypophysectomy was previously observed to allow high expression of CYP2B1 2 and 3A1 2 in the normally silent periportal region. In the present study, it had much less effect on the zonation of the ethanol-inducible P450 2E1 form: only a moderate shift of 2E1 staining towards the periportal region was observed by immunohistochemistry. Subsequent injections with T 3 moderately decreased CYP2E1 expression in the periportal region and no significant countereffect of GH was discerned. T 3 treatment, previously observed to block only the periportal expression of CYP3A1 2 , counteracted the increased CYP2B1 2 expression caused by hypophysectomy equally in the periportal and perivenous region. This was true both at the protein and mRNA level, as analysed from cell lysates obtained by in situ perfusion of livers by zone-restricted digitonin treatment. Thus, although hypophysectomy and subsequent GH and T 3 treatment affect the total expression of CYP2B1 2 , 2E1, and 3A1 2 similarly, the zonal effects were isozyme-specific. In contrast, the perivenous zonation normally seen for the dioxin-inducible P450 1A2 form was steepened rather than diminished by hypophysectomy, both in male and female rats. Administration of GH by the female-type continuous infusion had no effect in male rats, but partially counteracted the effect of hypophysectomy in females, suggesting an involvement of GH. In contrast to other CYP genes investigated, the female-characteristic expression of CYP2C12 was found to be completely non-zonated. Hypophysectomy and continuous GH administration dramatically affected the amount of mRNA of both P450 2C12 and the male-specific 2C11 form, but analysis of periportal and perivenous cell lysates indicated that these effects were not zone-specific. The distribution of the GH receptor was investigated to explain the zonal effects of GH. Immunohistochemically, a moderate perivenous dominance was observed, whereas the mRNA abundance of both GH receptor and GH binding protein was slightly higher in the periportal region. Thus, zonal regulation by GH does not appear to result from a GH receptor zonation; rather, a sinusoidal GH gradient may be involved. These data, combined with our previous results, indicate that pituitary-dependent hormones regulate the zone-specific expression of some P450 forms strongly (i.e. 2B1 2 and 3A1 2 ), and other forms are moderately regulated (i.e. 1A2 and 2E1), or are affected across the whole acinus (i.e. 2C11, 2C12).

Contrasting metabolic effects of continuous and pulsatile growth hormone administration in young adults with Type 1 (insulin-dependent) diabetes mellitus

Plasma growth hormone profiles in adolescents with Type 1 (insulin-dependent) diabetes mellitus are characterized by both increases in pulse amplitude and higher baseline concentrations. To determine which of these abnormalities adversely affect metabolic control, we studied six young adults overnight on three occasions. On each night somatostatin (50-100 micrograms.m2-1.h-1) and glucagon (1 ng.kg-1.min-1) were infused continuously and 18 mU/kg of growth hormone was given as either: three discrete pulses of 6 mU.kg-1.h-1 at 180-min intervals or a 12-h infusion (1.5 mU.kg-1.h-1) or buffer solution only on a control night. Euglycaemia was maintained by an insulin-varying clamp. Blood samples were taken every 15 min for glucose and growth hormone and every hour for intermediate metabolites and non-esterified fatty acids. Comparable normoglycaemic conditions were achieved on all three nights. Growth hormone levels achieved (mean +/- SEM) on study nights were: 32.8 +/- 2.2 mU/l (peak level during growth hormone pulses); 9.8 +/- 0.8 mU/l (continuous growth hormone) and 1.1 +/- 0.3 mU/l (control level). Pulsatile growth hormone administration led to an increase in insulin requirements (mean +/- SEM: 0.17 +/- 0.03 vs control 0.09 +/- 0.01 mU.kg-1.min-1, p less than 0.05) whereas insulin requirements following continuous growth hormone administration were unchanged. Cross-correlation confirmed an increase in insulin requirements occurring 135 min after a growth hormone pulse (r = 0.21, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Research Note: Hepatomegaly Induced by the Pulsatile, but not Continuous, Intravenous Administration of Purified Chicken Growth Hormone in Broiler Pullets: Liver Composition and Nucleic-Acid Content

The present study determined the composition and nucleic-acid content of livers obtained from 11-wk-old, broiler-strain pullets infused with pituitary-derived chicken growth hormone (p-cGH) in either a pulsatile (Study 1) or continuous (Study 2) manner for 21 days. Pulsatile p-cGH administration resulted in a decrease in ratios for liver DNA to wet weight (P<.01), DNA to protein (P<.03), and DNA to RNA (P<.002), compared with controls. Total RNA-to-liver ratio (P<.003) and liver weight (P<.007) were significantly elevated, compared with controls. Total grams of protein (P<.0007), water (P<.004), and ash (P<.01) were also elevated, with no significant differences in the percentage of composition. Continuous growth-hormone (GH) administration resulted in a decrease in the DNA-to-protein ratio (P<.04) and a modest increase in the mass of liver protein (P<.11) in comparison with controls, suggestive of cell hypertrophy. On a mass and percentage basis, liver composition was otherwise unaffected by the continuous administration of GH. In summary, pulsatile administration of GH induced hepatomegaly, largely due to cell hypertrophy, with no change in relative tissue composition. The continuous administration of GH had no effect on liver size, tissue composition, or most nucleic-acid-based indicators of cell hypertrophy or hyperplasia. Thus, previously observed differences in overall growth performance due to pattern of GH administration were also reflected in selected target-tissue response.

Structural and Regulatory Analysis of the Male-Specific Rat Liver Cytochrome P-450 g: Repression by Continuous Growth Hormone Administration.

Complementary DNA clones encoding the male-specific rat liver cytochrome P-450 g have been isolated by cross-hybridization with sequences from the female-specific rat liver cytochrome P-450 15 beta. Tissue distribution analysis indicates the liver as the organ with major expression of this cytochrome P-450 gene. Minimal P-450 g expression was also detected in prostate, kidney, heart, and brain. A developmental analysis reveals liver expression in the 8-week-old male and to a lesser extent in the 4-week-old male, but no detectable expression is seen in females of these ages or in 1- and 2-week-old rats from both sexes. Hypophysectomy of female rats dramatically increases hepatic expression of P-450 g, whereas continuous GH administration represses hepatic expression in male or female hypophysectomized rats. In similarity to P-450 15 beta and P-450 16 alpha, therefore, the cytochrome P-450 g gene in liver is GH regulated.

CDNA cloning, sequence, and regulation of a major female-specific and growth hormone-inducible rat liver cytochrome P-450 active in 15 beta-hydroxylation of steroid sulfates.

cDNA clones encompassing the complete coding sequence for the female-specific rat liver microsomal 15 beta-hydroxylase have been isolated and sequenced. This cytochrome P-450 species (P-450 15 beta) has a total of 490 amino acid residues, and its deduced amino acid sequence as well as direct sequencing of the amino-terminal portion of the purified protein revealed its identity with P-450 i. P-450 15 beta was found to be 66% similar to the male-specific rat liver microsomal 16 alpha-hydroxylase, P-450 16 alpha. Furthermore, P-450 15 beta is 47% similar to the major phenobarbital-inducible cytochrome P-450 in rat liver, P-450 b, while its structural similarity to P-450 c, P-450 pregnenolone-16a-carbonitrile, and P-450 lauric acid omega-hydroxylase is less than 30%. However, the developmentally regulated cytochromes P-450 f and P-450 PB-1 both have 68% similarity to P-450 15 beta. It is therefore concluded that P-450 15 beta belongs to the same gene subfamily (P450IIC) that also includes cytochromes P-450 f, PB-1, and 16 alpha. The plasma pattern of growth hormone (GH) is known to be a major determinant for the sex-specific expression of P-450 15 beta and P-450 16 alpha. Continuous infusion of GH to hypophysectomized male rats causes an increase in P-450 15 beta mRNA after 6 days of treatment, while intermittent injections have no effect. On the other hand, no effect on P-450 16 alpha mRNA is observed after continuous administration of GH, while intermittent injections cause an increase after 2 days of treatment. This relatively long period before P-450 15 beta and P-450 16 alpha mRNA induction is seen might indicate that protein factors mediating GH action are involved.

Induction of growth hormone (GH) receptors in adipocytes of hypophysectomized rats by GH.

The ability of GH to regulate its own receptor in adipocytes of hormone-substituted hypophysectomized rats was studied. Male rats (130-150 g) were hypophysectomized and substituted with T4 and cortisone. A fixed dose of GH (bovine GH, human GH, or ovine GH), was administered for 5-6 days in three different ways: 1) two injections/day, 2) four injections/day, or 3) by osmotic minipumps. GH binding was measured in cell aliquots using [125I]human GH. In unsubstituted hypophysectomized animals, GH binding was decreased and was approximately 25% of the binding observed in adipocytes of normal rats. With T4 and cortisone replacement, GH binding was partially restored. When GH was administered in four daily injections or via osmotic minipumps, a further increase in GH binding was observed. This increase was observed if the animals were killed up to 6 h but not 12 h after the last GH injection. GH given in two daily injections had no effect on GH binding even when studied at different time periods after the last GH injection. The GH receptor induced by frequent or continuous administration of GH was mainly somatogenic, since an excess of unlabeled ovine PRL inhibited GH binding only to a minor extent. There was no difference in accumulated body weight gain between the GH-treated groups during the treatment period. The results show that GH regulates its own receptor in adipocytes and that the mode of administration of the hormone is of importance for this effect of GH.

Limb regeneration following the discontinuation of growth hormone therapy in the hypophysectomized newt, Notophthalmus viridescens.

Untreated adult newts do not undergo normal limb regeneration following hypohysectomy. A fibrocellular dermal barrier (cicatrix) atypically forms between the apical epithelium and the underlying mesenchymal tissues. Historically, continuous administration of growth hormone or of prolactin in combination with thyroxine restored regenerative capacity to these newts. In a previous investigation, we demonstrated that the initial effect of these two hormone treatments, when administered on alternate days to hypophysectomized newts beginning eight days post-amputation, was to facilitate the erosion of the fibrocellular barrier and establish the epithelial mesenchymal interface that is observed in a regenerating limb. The present investigation was designed to evaluate the necessity of continuous hormone therapy to maintain limb regeneration in hypophysectomized newts. One, two, or three injections of growth hormone or of prolactin in combination with thyroxine was administered on successive alternate days to hypophysectomized newts either immediately following limb amputation (ID) or beginning eight days post-amputation (DD). The ID and DD newts receiving one, two, or three injections of growth hormone showed evidence of regeneration to the digitiform stage by day 30 post-amputation, while those receiving prolactin and thyroxine underwent wound healing. While both hormone treatments initially promoted a dermis-free apical epithelium, only hypophysectomized newts that had received growth hormone were able to continue regenerating. We have, therefore, concluded that discontinuous growth hormone therapy is sufficient to initiate and maintain the conducive environment for limb regeneration to advanced stages in the hypophysectomized newt. While initiating this process, prolactin and thyroxine therapy on a discontinuous regime does not maintain regeneration. The direct and indirect role of growth hormone in supporting limb regeneration in normal and hypophysectomized newts is discussed.

SUPRAHYPOPHYSEAL DYSFUNCTION IN A PATIENT WITH ASEXUAL ATELEIOSIS

The neuroendocrine function of a 19 years old female dwarf with primary amenorrhoea and lack of sexual development (asexual ateleiosis) was studied. Undetectable fasting plasma levels of growth hormone (GH) and a lack of response to three different provocative stimuli was observed. Oestrogen administratin did not modify the GH response. Thyroid and adrenal function were within normal limits. Undetectable plasma levels of immunoreactive oestradiol and lack of oestrogenic activity in vaginal smears indicated absence of ovarian function. Low levels of circulating gonadotrophins with a significant rise after synthetic LH-RH administration was demonstrated, while clomiphene citrate failed to induce ovulation. Following 6 months of continuous GH administration a significant increase in the growth rate was evident, whereas no pubertal development was observed. These data are interpreted as demonstrating the suprahypophyseal origin of the sexual infantilism in a patient with inappropriate GH secretion. It is suggested that a combined deficiency of LH-RH and GH-RH may account for the aetiology of this disorder.