Influence of growth hormone on bone marrow adipogenesis in hypophysectomized rats

The hypophysectomized rat has been used as a model to study the effects of growth hormone deficiency on bone. Here, we have investigated the influence of growth hormone administration to hypophysectomized rats (HX) for 6 wk on accumulation of triglycerides in bone marrow and on the differentiation of primary marrow stromal cells into adipocytes under in vitro conditions. We found that hypophysectomy significantly increased triglyceride concentration in bone marrow, which was attenuated by growth hormone administration. Primary bone marrow stromal cells derived from HX rats also had more adipocytes at confluence compared with growth hormone-treated hypophysectomized (GH) rats. When stimulated with 3-isobutyl-1-methylxanthine plus dexamethasone (IBMX-Dex), preadipocyte colony counts increased more significantly in GH rats. Markers of adipocyte differentiation were higher in HX than in control or GH rats at confluence. However, after stimulation with IBMX-Dex, increased expression of markers was seen in GH compared with HX rats. In conclusion, growth hormone administration to hypophysectomized rats attenuated triglyceride accumulation in bone marrow and inhibited the differentiation of stromal cells into adipocytes in vitro.     

Effects of corticotropin releasing factor on locomotor activity in hypophysectomized rats

Corticotropin releasing factor (CRF) injected intracerebroventricularly to hypophysectomized and sham hypophysectomized rats produced a dose dependent increase in locomotor activity, but in untreated hypophysectomized rats 10× more CRF was needed to produce a significant increase in activity. Concomitant daily supplements of rat growth hormone, thyroxine, and corticosterone to the hypophysectomized rats eliminated locomotor activity differences between the two groups. There was no statistically significant difference in locomotor response to either saline, 0.1 μg CRF, 1.0 μg CRF or 10.0 μg CRF in the group of animals receiving hormonal supplements. These results demonstrate that CRF can produce behavioral activation in rats independently of its effects on releasing hormones from the pituitary gland.     

Immunohistochemical analysis of EGF in epiphyseal growth plate from normal,hypophysectomized, and growth hormone-treated hypophysectomized rats

Epiphyseal growth plate cartilages from the proximal tibia of normal, hypophysectomized, and growth hormone (GH)-treated hypophysectomized rats were subjected to immunohistochemistry for detection of epidermal growth factor (EGF). In the normal growth plate, EGF was distributed mainly in the proliferative zone. Hypophysectomy resulted in considerable atrophy of the chondrocytes and the cartilage matrix (a decreased number of mature-type chondrocytes and a decreased ratio of proliferating to hypertrophic chondrocytes) and a significant diminution of EGF immunoreactivity. Treatment with GH reversed these effects of hypophysectomy, causing an increased thickness of the growth plate and EGF-reactive sites in all chondrocyte layers. The most intense immunostaining for EGF, however, was frequently seen in the nuclei of chondrocytes with flattened appearance. It appears that EGF could be incorporated or synthesized in chondrocytes having marked mitogenic activity. The present results, taken with previous data on EGF involvement in growth of cartilaginous tissue in vivo and in vitro, strongly suggest that EGF-immunoreactive chondrocytes are involved in cartilage proliferation and growth under the specific influence of GH.     

Regulation of insulin-like growth factor I binding in the rat uterus by growth hormone and thyroxine.

The insulin-like growth factor-I (IGF-I)-binding sites in the rat uterus were characterized further and the effects of growth hormone and thyroxine examined. The 125I-labelled IGF-I binding sites on uterine membranes demonstrated relative binding affinity of less than 20% for IGF-II, less than 1% for insulin and no affinity for an unrelated peptide, epidermal growth factor, compared with 100% for IGF-I confirming the specificity of these binding sites. Scatchard analysis of the specific binding data revealed the presence of a single class of high-affinity binding sites (Kd = 2.50 +/- 0.68 nmol l-1, with a binding capacity of 1.02 +/- 0.13 pmol mg-1 membrane protein in the uterus of the pituitary-intact ovariectomized rat. After hypophysectomy, the uteri from these rats had significantly (P less than 0.05) increased IGF-I binding sites, without significant changes in their affinity. Administration of growth hormone with or without L-thyroxine reversed this increase in IGF-I binding. Injection of thyroxine alone to the hypophysectomized ovariectomized rats had no significant effects on the uterine IGF-I binding sites. These data show that growth hormone, but not thyroxine, can regulate IGF-I binding sites in the rat uterus, possibly through regulating IGF-I production.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

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.     

Effect of mammalian growth hormone and prolactin on the growth of hypophysectomized chickens

Body weight gain and shank-toe growth during a 26-day treatment period following hypophysectomy were 55 and 46%, respectively, of control values, but the body weight gain was unaffected and bone growth only slightly reduced when the hypophysectomized chickens were fed a low dose of corticosterone (5 ppm). Bovine growth hormone (0.5 mg GH/kg body wt/day for 18 days) enhanced body weight gain and shank-toe length increase (an estimate of bone growth) by 46 and 33%, respectively, compared to the growth of hypophysectomized chickens receiving only corticosterone. These same endpoints were increased approximately 24% after ovine growth hormone treatment in hypophysectomized chickens not receiving corticosterone. Body weight gain during 18 days of treatment with bovine prolactin (0.5 mg PRL/kg/day) was 27% greater than the value for corticosterone-treated hypophysectomized chickens, but bone growth was unaffected. The mammalian GH preparations increased heart weight of the hypophysectomized chickens (25-29%), but pectoralis muscle weight was unaffected. GH treatment enhanced thymal weights by 71% in corticosterone-treated hypophysectomized chickens, and by 93% in hypophysectomized animals not receiving corticosterone. GH had no significant effect on bursal weights, and PRL had no effect on either of these lymphoid organ weights in corticosterone-treated hypophysectomized chickens. GH increased liver and adipose tissue weights considerably more than the large increases that followed treatment of hypophysectomized chickens with corticosterone alone (69 and 126% greater, respectively), but had no effect on these endpoints in hypophysectomized chickens not receiving corticosterone. PRL also greatly increased liver and adipose tissue weights in corticosterone-treated hypophysectomized chickens (79 and 75%, respectively). These results provide evidence that mammalian GH enhances body weight gain, bone growth, and the growth of several organs in the hypophysectomized chicken. Mammalian PRL increased body weight gain, liver weight, and adipose tissue weight in corticosterone-treated hypophysectomized chickens, but did not influence bone growth or the weights of the heart, pectoralis, thymi, or bursa.     

Effect of growth hormone on fatty acid oxidation: growth hormone increases the activity of 2,4-dienoyl-CoA reductase in mitochondria

The effect of growth hormone on the beta-oxidation of saturated and unsaturated fatty acids was studied with mitochondria isolated from control rats, hypophysectomized rats, and hypophysectomized rats treated with growth hormone. Rates of respiration supported by polyunsaturated fatty acylcarnitines, in contrast to rates observed with palmitoylcarnitine or oleoylcarnitine, were slightly lower in hypophysectomized rats than in normal rats, but were higher in hypophysectomized rats treated with growth hormone. The effects were most pronounced with docosahexaenoylcarnitine, the substrate with the highest degree of unsaturation. Since uncoupling of mitochondria with 2,4-dinitrophenol resulted in lower rates of docosahexaenoylcarnitine-supported respiration, while substitution of ATP for ADP yielded higher rates, it appears that energy is required for the effective oxidation of polyunsaturated fatty acids. Growth hormone treatment of hypophysectomized rats caused a threefold increase in the activity of 2,4-dienoyl-CoA reductase or 4-enoyl-CoA reductase (EC 1.3.1.34) in mitochondria, but not in peroxisomes. The activities of other beta-oxidation enzymes remained virtually unchanged. Rates of acetoacetate formation from linolenoylcarnitine, but not from palmitoylcarnitine, were stimulated by glutamate in mitochondria from hypophysectomized rats and hypophysectomized rats treated with growth hormone. All data together lead to the conclusion that the mitochondrial oxidation of highly polyunsaturated fatty acids is limited by the availability of NADPH and the activity of 2,4-dienoyl-CoA reductase which is induced by growth hormone treatment.     

Influence of hormones on the nicotinamide nucleotide coenzymes of adipose tissue

The concentrations of the oxidized and reduced forms of the nicotinamide nucleotides were measured in the epididymal fat pads of normal, alloxan-diabetic and hypophysectomized rats. In both alloxan-diabetic rats and hypophysectomized rats the weight of the adipose tissue fell, as did the total content of NADH and NADPH; in addition, NAD⁺ was decreased in the alloxan-diabetic group. Of these changes the most marked was in NADPH and this was the only significant difference when the results were expressed as nicotinamide nucleotides/mg. of tissue protein. The concentration of NADPH in the hypophysectomized rats was not altered by treatment with growth hormone but was restored to normal by treatment with thyroxine. These results are discussed in relation to the known effect of these hormonal conditions on lipid synthesis in adipose tissue.     

Interacting role of thyroxine and growth hormone in the hepatic synthesis of alpha 2u-globulin and its messenger RNA

Hypophysectomy completely abolishes and thyroidectomy results in a 90% reduction in the hepatic content of alpha 2u-globulin and its mRNA in the male rat. Thyroid hormone is also known to be required for the synthesis and secretion of pituitary growth hormone. In the hypothyroid rat either thyroxine or growth hormone was found to increase the activity and number of sequences of the mRNA for alpha 2u-globulin (measured by translational assay and hybridizational analysis with a cloned cDNA probe) to the euthyroid level. Treatment of hypophysectomized rats with a hormone combination containing growth hormone but not thyroxine increased the hepatic level of the mRNA for alpha 2u-globulin to that of normal animals. From these results we conclude that thyroxine indirectly influences the hepatic concentration of the mRNA for alpha 2u-globulin through its effect on pituitary growth hormone. Although administration of growth hormone to hypothyroid animals raised the hepatic concentration of alpha 2u-globulin mRNA to the euthyroid level, synthesis of alpha 2u-globulin remained low (50% of the normal). Complete recovery of alpha 2u-globulin synthesis required thyroxine. Therefore, in addition to an indirect effect on the hepatic level of alpha 2u-globulin mRNA, thyroxine also directly influences the synthesis of this protein. This direct effect of thyroxine on alpha 2u-globulin synthesis seems to be exerted at a step distal to the formation of mature mRNA.     

An investigation of the prolactin-thyroxine synergism in newt limb regeneration

The use of hormone replacement to support limb regeneration in hypophysectomized newts has been the subject of many investigations. Growth hormone, as well as prolactin (PL) in combination with exogenously supplied thyroxine, have all been shown to he effective. However, the bovine growth hormone used to support limb regeneration was contaminated by prolactin and thyroidstimulating hormone (TSH). The present investigation evaluates the significance of (1) prolactin contamination and (2) endogenous thyroxine synthesis resulting from TSH contamination on limb regeneration in hypophysectomized newts. The effect of supplying exogenous thyroxine was also evaluated. Our studies showed that when hypophysectomized newts were injected with contamination levels of PL and TSH, regeneration occurred, suggesting that the newt's thyroid synthesized sufficient thyroxine to support a prolactin-thyroxine synergism. The endogenous thyroxine was synthesized by thyroid glands that were indistinguishable from those of saline-injected, hypophysectomized controls.     

Influence of growth hormone and thyroxine on thermotropic effects of respiration and 1-anilino-8-naphthalene sulfonate fluorescence and on lipid composition of cardiac membranes

The effects of hypophysectomy and subsequent administration of growth hormone and/or L-thyroxine on thermotropic properties of State 3 respiration (ADP-induced), cholesterol, phospholipid and fatty acid composition of phospholipid fraction were examined in myocardial mitochondria of rats. Temperature-dependence of 1-anilino-8-naphthalene sulfonate fluorescence was determined in vesicles prepared from lipids of heart mitochondria. Transition temperature obtained from the Arrhenius plots of respiration occurred at 21 and 24°C for heart mitochondria of normal and hypophysectomized rats, respectively. Most notably, after hypophysectomy the rate of respiration was lower below 24°C, but was progressively higher above that temperature when compared to normal rats. The energy of activation was 148 and 36% larger below and above the transition temperature, respectively. Growth hormone restored almost completely the energy of activation and respiratory rates to normal levels. Administration of L-thyroxine, with or without growth hormone, did not significantly change the rate of respiration but decreased the transition temperature to 17.7–17.0°C. Lipid and phospholipid content, as well as percent distribution of phospholipids and their fatty acid composition were not statistically different among the different groups of rats. Only cholesterol content was increased after hypophysectomy. Administration of growth hormone and thyroxine did not significantly change the total unsaturation index of fatty acids, but growth hormone increased the content of arachidonic acid (20 : 4) by 70% but decreased the docosahexaenoic acid (22 : 6) three times which may have a beneficial effect on mitochondrial membranes. These and other results suggest that hormones exert different effects on subcellular organelles in different tissues, like heart and liver.     

Endocrine regulation of the growth plate

Longitudinal bone growth occurs at the growth plate by endochondral ossification. Within the growth plate, chondrocyte proliferation, hypertrophy, and cartilage matrix secretion result in chondrogenesis. The newly formed cartilage is invaded by blood vessels and bone cells that remodel the newly formed cartilage into bone tissue. This process of longitudinal bone growth is governed by a complex network of endocrine signals, including growth hormone, insulin-like growth factor I, glucocorticoid, thyroid hormone, estrogen, androgen, vitamin D, and leptin. Many of these signals regulate growth plate function, both by acting locally on growth plate chondrocytes and also indirectly by modulating other endocrine signals in the network. Some of the local effects of hormones are mediated by changes in paracrine factors that control chondrocyte proliferation and differentiation. Many human skeletal growth disorders are caused by abnormalities in the endocrine regulation of the growth plate. This review provides an overview of the endocrine signals that regulate longitudinal bone growth, their interactions, and the mechanisms by which they affect growth plate chondrogenesis.     

Influence of thyroxine and luteinizing hormone on some enzymes concerned with lipogenesis in adipose tissue, testis and adrenal gland

The activities of hexokinase, citrate-cleavage enzyme, ;malic enzyme' and NADP-linked isocitrate dehydrogenase have been measured in the adipose tissue, testes and adrenals of normal rats, hypophysectomized rats and hypophysectomized rats treated with either thyroxine or thyroxine plus luteinizing hormone. Hypophysectomy reduced the activity of all four enzymes in all three tissues. Thyroxine alone restored the activity of all four enzymes in adipose tissue towards normal but failed to do so in either testes or adrenals. Thyroxine and luteinizing hormone restored the citrate-cleavage enzyme activity of testes and increased the activity of hexokinase from the low value after hypophysectomy. Neither ;malic enzyme' nor isocitrate dehydrogenase was increased by thyroxine or thyroxine and luteinizing hormone in testes. The differential stimulation of enzyme activity by thyroxine in the different tissues suggests thyroxine as having a special significance in adipose-tissue lipogenesis.     

Stimulation of ornithine decarboxylase activity in rat tissues by growth hormone and by serum growth factor from rats infested with spargana of Spirometra mansonoides

Ornithine decarboxylase activity was studied in heart, kidney, liver, thymus, lung, spleen, skeletal muscle and fat of hypophysectomized rats after growth hormone treatment. A marked increase in enzyme activity was observed in kidney and liver, and a significant increase in heart and thymus at 4 h after injection of growth hormone. The kidney was the most responsive organ with an increase in activity of about 100 fold. The enzyme activity in kidney responded to a dose of 10 μg of growth hormone. Daily injection for 12 days raised activity only in the heart. Infestation for 6–13 days with spargana of Spirometra mansonoides, which also causes growth of hypophysectomized rats, increased enzyme activity in the heart and thymus. Intravenous injection of serum of hypophysectomized rats infested with spargana of Spirometra mansonoides caused a significant increase in the enzyme activity in liver and kidney after 4 h. Growth hormone and the serum growth factor of sparganosis seem to share the characteristic of causing an early increase in ornithine decarboxylase activity in rat tissues. The marked response in kidney and liver raises the possibility that these organs are the primary targets of both substances.     

Effects of hypophysectomy, growth hormone, and thyroxine on protein turnover in heart.

Cardiac atrophy following hypophysectomy was accompanied by decreased heart content of RNA and polysomes and increased levels of ribosomal subunits, suggesting that protein synthesis was restricted by a reduced supply of ribosomes and an imbalance between rates of peptide-chain initiation and elongation. During perfusion in vitro, provision of palmitate restored the normal balance between rates of initiation and elongation but protein synthesis was lower in hearts of hypophysectomized than normal rats, reflecting the lower RNA content of hearts from hormone-deficient animals. After the period of atrophy had passed, or after treatment with growth hormone and thyroxine, heart RNA content and rates of protein synthesis were equal to or greater than those found in normal hearts. When plasma levels of amino acids, glucose, fatty acids, and insulin, and rates of beating and ventricular pressure development observed in normal and hypophysectomized rats were simulated during in vitro perfusion, hearts from hormone-deficient rats had reduced rates of protein synthesis but unaltered rates of degradation. Cathepsin D activity in heart homogenates (+ Triton X-100) was elevated during cardiac atrophy when expressed per g of tissue but not when expressed per heart.     

Temporal analysis of rat growth plates: cessation of growth with age despite presence of a physis.

Despite the continued presence of growth plates in aged rats, longitudinal growth no longer occurs. The aims of this study were to understand the reasons for the cessation of growth. We studied the growth plates of femurs and tibiae in Wistar rats aged 62-80 weeks and compared these with the corresponding growth plates from rats aged 2-16 weeks. During skeletal growth, the heights of the plates, especially that of the hypertrophic zone, reflected the rate of bone growth. During the period of decelerating growth, it was the loss of large hydrated chondrocytes that contributed most to the overall decrease in the heights of the growth plates. In the old rats we identified four categories of growth plate morphology that were not present in the growth plates of younger rats: (a). formation of a bone band parallel to the metaphyseal edge of the growth plate, which effectively sealed that edge; (b). extensive areas of acellularity, which were resistant to resorption and/or remodeling; (c). extensive remodeling and bone formation within cellular regions of the growth plate; and (d). direct bone formation by former growth plate chondrocytes. These processes, together with a loss of synchrony across the plate, would prevent further longitudinal expansion of the growth plate despite continued sporadic proliferation of chondrocytes.