Presence Of hGH Research Articles

Gastric uptake of recombinant growth hormone in rainbow trout

The aim of the present study was to investigate gastrointestinal uptake of recombinant growth hormone in rainbow trout. Recombinant human GH was used in this study to avoid cross reactivity with the endogenous trout GH molecule. Using both in vitro and in vivo approach, the results demonstrate that stomach is the main site of uptake for orally administered intact recombinant human growth hormone (hGH) in rainbow trout. The presence of hGH was verified by western blot analysis in the stomach tissue as well as in the serum after 30 minutes of intubation. No traces of the hGH could be detected after 90 minutes of administration, indicating rapid clearance of the hormone in rainbow trout. Furthermore, no immunoreactive hGH was detected in other tissues including mid-gut, muscle and liver at the time points tested. The findings provide novel information on the gastric uptake of hGH in rainbow trout, and would be of relevance to the application of GH in aquaculture.

The D152H mutation found in growth hormone insensitivity syndrome impairs expression and function of human growth hormone receptor but is silent in rat receptor

In two patients with growth hormone (GH) insensitivity syndrome (Laron syndrome), in whom the GH receptor is able to bind the hormone, the D152H mutation was identified, and lack of dimerization was proposed to explain GH resistance in these patients. To examine further the consequences of the substitution of conserved aspartate 152 on the function of the GH receptor (GHR), we reproduced the mutation in vitro on the full length GH receptor cDNA from man and rat. Effects of the mutation on expression and activity of the GHR were analyzed in 293 cells transfected with wild-type and mutant GHR cDNAs. Mutant human receptor protein was expressed at a lower level than wild-type receptor and its activity was reduced: GH-dependent signal transducer and activator of transcription 5 (Stat5)-mediated transactivation of a reporter gene was lower in 293 cells transfected with mutant GHR cDNA than in transfected cells expressing a comparable level of wild-type GHR. The membrane-bound form of the mutant and of the wild-type human GHR were able to homodimerize, as suggested by the size of the complexes detected in cross-linking experiments with 125I-human (h) GH, and also by the activity in the functional test. With the soluble GHR resulting from proteolysis of the wild-type membrane form, no dimeric complexes could be detected. However, when a soluble receptor lacking the transmembrane and cytoplasmic domains of the receptor was expressed, wild-type and not mutant GH binding protein (GHBP) was able to form dimers in the presence of hGH. The amino acid substitution has no effect on either expression or function of the rat receptor. Structural modeling of D152H soluble human and rat GHR (GHBP) supports the species-specific functional consequences of the mutation. Evaluation of the functional importance of the mutation strongly suggests that impairment in expression and activity of the mutant receptor, rather than complete lack of dimerization, explains the GH resistance of the patients.

Growth hormone stimulates multinucleated cell formation in long-term bone marrow cultures

Although the effects of growth hormone on bone metabolism are well-documented, their role in the regulation of immune responses such as the inflammatory process has not been thoroughly explored. This study investigated the formation of multinucleated cells (MNC) in long-term human bone marrow cultures. Experiments using 1 and 100 ng/ml of human recombinant growth hormone (hGH) and 10(-7) M of 1,25 dihydroxyvitamin D3 (VD3) showed that hGH increased the total number and nucleation of MNC. The effects of hGH were generally greater than those observed with VD3. Cytological and immunological characterization of MNC revealed several macrophage polykaryon features. MNC did not respond to calcitonin in a cyclic adenosine monophosphate assay and failed to resorb dentin slices. These results demonstrate that MNC formed in the presence of hGH and VD3 present an essentially macrophage polykaryon phenotype. In this context, growth hormone may be involved in the inflammatory process through upmodulation of macrophage polykaryon formation.

Growth hormone regulates ammoniagenesis in canine renal proximal tubule segments.

To determine whether growth hormone (GH) directly affects ammoniagenesis in the renal proximal tubule, ammonia production was measured in suspensions of isolated canine renal proximal tubule segments (IPTs) incubated with 2.5 mM L-glutamine and varying concentrations of human growth hormone (hGH). Ammonia production from IPTs significantly increased by nearly threefold in the presence of hGH (10(-6) M) at 60 min. This increase was dose dependent, with as little as 10(-9) M hGH significantly stimulating ammonia production. In addition, hGH enhanced glucose production when lactate, alanine, and succinate replaced L-glutamine as substrate. hGH significantly stimulated ammonia production when IPTs were incubated at alkalotic and neutral pH. The effect of hGH was lost at acidic pH. When hGH was added to IPTs incubated under Na(+)-equilibrated conditions, ammonia production was not different from control. hGH stimulated ouabain-sensitive Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity by 8.1 +/- 1.1% in basolateral membranes isolated from IPTs. hGH stimulation of proximal tubule ammonia production from L-glutamine occurs at physiological concentrations of hGH and when the extracellular-to-intracellular Na+ gradient favors L-glutamine transport. This effect is associated with an increase in basolateral Na(+)-K(+)-ATPase activity. The data suggest a role for hGH in the regulation of renal acid-base metabolism under physiological conditions in which increased net acid excretion is important.

Biochemical and cytochemical studies of preadipocyte differentiation in serum-free culture of porcine stromal-vascular cells: interaction of dexamethasone and growth hormone.

Stromal-vascular cells from adipose tissue of pigs 5-7 days of age were grown in serum for 2-3 days and switched to serum-free (insulin, transferrin and selenium) conditions +/- test hormones for 6-7 days. The interaction of dexamethasone (DEX) and human growth hormone (hGH) was evaluated since glucocorticoids augment and hGH antagonizes the effect of insulin. Low levels (1-10 nM) of DEX with insulin doubled (p less than 0.05) specific activity of glycerol phosphate dehydrogenase (GPDH) and doubled (p less than 0.05) the number of detectable fat cells relative to insulin alone. DEX with insulin enhanced the morphological differentiation of preadipocytes and markedly increased fat cell cluster numbers in the presence of hGH. Furthermore, 1-10 nM of DEX partially blocked (p greater than 0.05) the inhibitory effect of 10 nM hGH on GPDH activity, but 1-100 nM DEX had no effect (p greater than 0.05) on the ability of hGH to compromise lipid deposition. DEX alone (no insulin or hGH) induced the appearance of esterase-reactive but lipid-free cells. Cells with these characteristics were increased in number by DEX in the presence of hGH but were nearly absent in the presence of insulin and DEX. Therefore, transient exposure to GH in vivo may have no permanent effect on adipose tissue development in the continued presence of glucocorticoids.

Human growth hormone and insulin-like growth factor-1 enhance the proliferation of human leukemic blasts.

As the number of long-term survivors of childhood leukemia increases, growth retardation has emerged as a significant complication. Treatment of these children with growth hormone (GH) has been suggested and sporadically implemented. We, therefore, studied the effect of human GH (hGH) and its by-product insulin-like growth factor-1 (IGF-1) on the growth of leukemic cells in vitro. Under serum-free conditions hGH and IGF-1 induced a significant dose-dependent proliferative effect on promyelocytic leukemia (HL60) and Burkitt's lymphoma (Daudi) cell lines. Anti-hGH antibodies negated the stimulatory effect of hGH and anti-IGF-1 serum abrogated the growth-promoting effect enhanced by IGF-1. Similar statistically significant stimulatory properties were found when freshly obtained marrow cells from four of five acute lymphoblastic leukemia (ALL) of childhood and four acute myelogenous leukemia (AML) patients were studied in ALL and AML blast-cell clonogenic assays. ALL colonies increased numerically by 72% (P less than .025) and AML colonies by 92% (P less than .01) in the presence of hGH at concentrations of 2.5 x 10(2) and 3.0 x 10(2) ng/mL, respectively. IGF-1 stimulated ALL and AML blast-colony growth at concentrations ranging from 0.05 to 0.5 ng/mL by up to 105% (P less than .025) and 65% (P less than .03), respectively. Our in vitro data suggest that circulating hGH and IGF-1 may promote leukemic blast cell replication in vivo, and the supplemental administration of hGH to leukemia patients in remission must be carefully monitored for early relapse.

37 GROWTH HORMONE HASTENS BONE MATURATION DURING ‘PUBERTY’ IN HYPOPITUITARY BOYS

Skeletal maturation rate (SMR) during spontaneous or induced (pseudo-) puberty was studied in boys with hypopituitary dwarfism, 30 treated with hGH, 26 without. Bone age (BA, Greulich & Pyle) was estimated at the time when pubertal signs were first noted or when androgen substitution was begun. When final height was reached pubertal growth was considered to have ended and BA assumed to be maximal (19 y). SMR (mean 0.91, SEM 0.058) was negatively correlated with initial BA (mean 11.2, SEM 0.37) in boys without hGH; in only 4/26 SMR exceeded 1 y/y. For boys with hGH there was no correlation between SMR (mean 1.54, SEM 0.070) and initial BA (mean 11.5, SEM 0.26); in 26/30 SMR exceeded 1 y/y.Conclusions: androgen-induced skeletal maturation occurs faster in presence of hGH than in its absence; this explains earlier finding of similar final heights of hypopituitary boys with and without hGH-treatment. Hence hGH-treatment must aim at greatest possible height before puberty.

TREATMENT WITH hGH HAS NO EFFECT ON ADULT HEIGHT IN HYPOPITUITARY CHILDREN

Adult height of hypopituitary children following treatment with human pituitary growth hormone (hGH) is disappointingly low despite the fact that hGH administration before puberty results in accelerated statural growth without unduly accelerating bone maturation. Our study showed that the range of adult heights was similar for various types of hypopituitary growth retardation; this range did not undergo an appreciable upward shift after hGH treatment. Evaluation of hGH effectiveness for a well-defined type, that of multiple pituitary hormone deficiency following breech birth, disclosed that adult height was not dependent on the presence of hGH (14 patients with hGH averaged 162.9 cm (SEM 1.93), 10 patients without hGH 159.5 cm (SEM 3.49)). However, adult heights were reached at earlier ages (with hGH at 22.3 y (SEM .38), without hGH at 25.7 y (SEM .54)). The duration of the pubertal spurt in growth brought about by androgen treatment was reduced by hGH from 9.5 y (SEM .91) to 5.5 y (SEM .45). Skeletal maturation must have been faster during androgen with hGH than during androgen alone. More detailed follow-up studies are needed, particularly in view of the imminent rise in the number of patients that will undergo treatment with biosynthetic growth hormone. Earlier treatment with higher or more frequent doses may be required to improve effectiveness of GH on adult stature.

Growth hormone potentiates colony formation of epiphyseal chondrocytes in suspension culture.

The effect of human GH (hGH) on colony formation of rat epiphyseal plate chondrocytes was studied in suspension culture. Chondrocytes were isolated enzymatically from epiphyseal plates of the proximal tibia of 28-day-old normal male rats, and were cultured in a suspension stabilized with 0.5% agarose. After approximately 7 days of culture in the presence of 10% newborn calf serum (NCS), chondrocyte colonies developed consisting of varying numbers of cells in matrix. No colonies developed in the absence of NCS, and the number of formed colonies was proportional to the concentration of NCS (5-20%) in the medium. hGH potentiated the formation of large size colonies (diameter greater than 90 microns) after a culture period of 10 or 14 days. The lowest effective concentration of hGH was 10 ng/ml, while 40 ng/ml hGH gave a maximal stimulatory effect (40-50%). Higher concentrations of hGH (80 and 160 ng/ml) showed reduced potentiation of colony formation. The stimulatory effect of hGH was expressed at 10-15% of NCS at 14 days of culture. There was a linear relation between the number of seeded cells and the number of colonies formed, both in the absence and presence of hGH. These results show that GH potentiates colony formation in chondrocytes of the epiphyseal growth plate, providing further support for the contention that GH exert a direct stimulatory effect on epiphyseal cartilage and thus stimulates longitudinal bone growth directly. The finding that GH preferentially potentiated the formation of large size colonies suggests that GH promoted the differentiation of early proliferative chondrocytes or stem cell chondrocytes with an inherent high capacity to proliferate.

Transplantation of human pituitary adenomas into nude mice.

In vitro studies with human pituitary adenomas are limited by the small amount of tissue obtained, which may be contaminated by the surrounding normal pituitary tissue. In this study we investigated if the passage of adenoma tissue via thymusless nude mice could solve some of these problems. The secretory capacity of the transplanted human pituitary adenomas was demonstrated by the presence of hGH and/or hPRL in the plasma of the host mouse, while other human pituitary hormones (TSH, LH and FSH) were undetectable. The transplants, however, decreased in size with time although histologically viable adenoma tissue was recovered that resembled the original tumor. Upon trypsinization of the small tissue fragments no viable adenoma cells could be obtained. In contrast, an experimental malignant rat pituitary tumor grew steadily with time, resulting in high levels of rPRL in the nude mice recipients. Large numbers of viable tumor cells were recovered from these tumors. Thus, human pituitary adenomas transplanted in nude mice continue to release hormone(s), but the transplants decrease in size and cannot be used to isolate dispersed tumor cells for in vitro studies.

The effect of growth hormone on the Leydig cell response to chorionic gonadotrophin in boys with hypopituitarism.

Eleven boys with growth hormone (hGH) deficiency received human chorionic gonadotrophin (hCG) stimulation tests for the assessment of Leydig cell function before, during, and after 1 year of treatment with somatotrophin. Two patients entered puberty during the course of the study protocol. Analysis of the data in nine prepubertal boys revealed an augmentation of testosterone (T) responses to hCG in the presence of hGH. In six of these individuals in whom dihydrotestosterone (DHT) was determined, a similar augmentation in responsiveness of this steroid was found in the presence of hGH. Three prepubertal boys exhibited poor T responses to the basal hCG test with only partial improvement following hGH. In man growth hormone may be an important permissive factor in Leydig cell activity during periods of changing testicular function such as occur in utero or during puberty.

Characterization of the slowly dissociable human growth hormone binding component of isolated rat hepatocytes.

Human growth hormone (hGH) bound to specific sites on rat hepatocytes. The time course of hGH dissociation was comprised of more than one component. Dissociation was resolved into rapid (t1/2 = 10.5 min) and slow (t 1/2 = 6.4 h) fractions. The amount of slowly dissociable hormone increased for the first 75 min during which time cells and [125I]hGH associated. Subsequently, the amount of slowly dissociable hGH was constant. The time courses of hGH receptor binding and subsequent retention of slowly dissociable label were similar. The capacity of hepatocytes to accumulate slowly dissociable label was saturated by hGH over the same concentration range as the high-affinity binding site (KD approximately 2 nM). This suggested that a receptor-mediated process was responsible for the accumulation of slowly dissociable hGH. Rapidly dissociable label was intact [125I]hGH and fragments resulting from growth hormone degradation. Slowly dissociable hGH recovered from hepatocytes by acid extraction was intact and immunocompetent. There was a large increase in the extent of [125I]hGH degradation between 23 and 37 degrees C. Over this temperature range, the proportion of hGH not in rapid equilibrium with the medium decreased. High concentrations of hGH decreased the amount of slowly dissociable [125I]hGH retained by hepatocytes by competing for high-affinity sites. The interaction of [125I]hGH with low-affinity degradative systems was favored by the presence of hGH. The temperature and concentration dependencies of hGH retention and degradation distinguished these proceses.

Growth and mitotic rate of the proximal tibial epiphyseal plate in hypophysectomized rats given estradiol and human growth hormone.

Hypophysectomized female immature albino rats were injected with HGH, HGH and estradiol, or estradiol solely. Some animals were left as controls. All animals were killed on day 3 or 5 of the experiment. One hour before sacrifice, the animals were flash labelled with tritiated thymidine. The following parameters were registered: labelling index, thickness of the growth plate, weight gain, and size of the uterus. The following conclusions were drawn. 1. Estradiol decreases the mitotic rate and retards growth of the epiphyseal cartilage. Its effect on the mitotic rate is more pronounced than its effect on the thickness of the tibial epiphyseal plate. 2. HGH increases the mitotic rate and promotes growth of the epiphyseal cartilage. Its effect on the thickness of the epiphyseal plate is more pronounced than its effect on the mitotic rate. 3. Estradiol decreases the mitotic rate even in the presence of HGH. 4. Estradiol seems to have a direct effect on mitosis of the chondrocytes.