Preservation of bone mass in hypogonadal female monkeys with recombinant human growth hormone administration.

This study examined the effect of human recombinant GH supplementation on bone loss in female monkeys made hypogonadal with GnRH agonist (GnRH-Ag). Animals were randomly assigned to three treatment groups: vehicle, GnRH-Ag, and GnRH-Ag and GH. After an initial 5-month pretreatment period during which all animals were maintained on a normal monkey chow diet containing a high level of calcium (1%) animals were maintained on a normal monkey chow diet containing a high level of calcium (1%), animals were shifted to a lower calcium diet (0.1%) for 4 to 5 months before the beginning of treatment and were maintained on this diet throughout the remainder of the study. Monkeys were treated continuously for 10 months with 25 micrograms/day GnRH-Ag or vehicle. GH was administered by im injection three times per week at a dose of 100 micrograms/kg body wt/day. Animals treated with GnRH-Ag were amenorrheic throughout the treatment period, and serum estradiol and progesterone levels were below minimum levels of detection. Vehicle-treated animals continued to cycle throughout the study. Monkeys treated with GnRH-Ag alone showed a significant decline (12%) in bone mineral density (BMD) of the lumbar spine. BMD was reduced below pretreatment levels from 6 months of GnRH-Ag treatment through 3 months post treatment in this group. GH supplementation reduced the decline of BMD in GnRH-Ag-treated monkeys. BMD did not change significantly with time in the GH-supplemented group. BMD values in GH supplemented animals between 5 and 10 months of the treatment period exceeded levels in animals treated with GnRH-Ag alone, but BMD levels during this interval were lower than in the vehicle-treated group. In the vehicle-treated group, there was small, but significant, increase in BMD over the course of the study. Serum osteocalcin concentrations were elevated above pretreatment values after 6 and 9 months of GnRH-Ag treatment alone or with GH supplementation, but did not change in vehicle-treated animals. GH also increased serum insulin-like growth factor 1 levels. In response to the lower calcium diet, serum PTH levels increased approximately 200% in vehicle-treated monkeys and animals treated with GnRH-Ag alone. GH attenuated this increase in serum PTH. The data indicate that the level of calcium in the diet of adult monkeys can be reduced more than 10-fold without affecting lumbar BMD provided ovarian function is normal, but if animals are made hypogonadal with a GnRH-Ag, bone mass declines.(ABSTRACT TRUNCATED AT 400 WORDS)

Failure of growth hormone to alter the biomechanics of fracture-healing in a rabbit model.

Standardized tibial osteotomies were created and stabilized with external fixation in twenty-seven skeletally mature rabbits. Fourteen animals were treated with a daily injection of human growth hormone (150 micrograms per kilogram of body weight), and thirteen received a daily injection of saline solution. Serial non-destructive biomechanical tests, radiography, and determinations of the levels of serum insulin-like growth-factor I were performed for all of the animals. Destructive strength-testing of the sites of osteotomy was performed at four, six, or eight weeks. Twenty-five of the twenty-seven osteotomies healed uneventfully. There were no significant differences in the serial biomechanical measurements at the sites of the healing osteotomies, in the radiographic measurements, or in the ultimate strength of the sites of the osteotomy between the two groups. The mean level of serum insulin-like growth-factor I increased 33 per cent relative to the preoperative baseline level in the group that received growth hormone and increased 10 per cent in the control group. This difference was not statistically significant. There was no significant correlation between the biomechanical properties at the sites of the osteotomies and the levels of serum insulin-like growth-factor I. Administration of growth hormone had no measurable effect on fracture-healing in this model of normal animals. It remains to be determined whether injection of growth hormone might affect healing when there is a state of deficiency of endogenous growth hormone or when there is a non-union of a fracture.

Effects of growth hormone on neonatal growth in nursing rhesus monkeys.

The effects of recombinant human GH treatment of either nursing mothers or their infants on neonatal growth in rhesus monkeys was determined. Growth rates of infants treated daily from birth with GH (INFGH; n = 9; 100 micrograms/kg, sc) were compared to those of infants given saline (INFc; n = 10), infants whose mothers received saline from the second trimester of pregnancy through 7 weeks postpartum (CON; n = 9), infants of mothers who received GH during pregnancy only from the second trimester to parturition (PRG; n = 8), infants of mothers who received GH during lactation only from parturition through 7 weeks postpartum (LAC; n = 9), and infants of mothers who received GH during the second trimester of pregnancy through 7 weeks postpartum (PRG/LAC; n = 8). Mothers receiving GH were given 250 micrograms/kg, sc, Monday, Wednesday, and Friday. Infants were allowed to nurse ad libitum. Although infant birth weights were similar among the six groups, body weights at 7 weeks of age were significantly greater in PRG/LAC infants (0.77 +/- 0.03 kg) compared to those in CON (0.66 +/- 0.02 kg), INFc (0.62 +/- 0.03 kg), LAC (0.62 +/- 0.04 kg), and INFGH infants (0.62 +/- 0.01 kg), with infants of PRG mothers intermediate (0.71 +/- 0.02 kg) between them. By 35 weeks of age, after infants had been weaned by their mothers, body weights were similar among all groups. Serum concentrations of insulin-like growth factor-I (IGF-I) rose significantly in all infants during the study period. Although IGF-I levels did not vary significantly among the treatment groups, average concentrations of IGF-I were significantly related to weight gains. Analyses of milk composition revealed that total protein, lactose, and IGF-I levels were similar among groups, whereas the percentage of fat in the milk was significantly higher in PRG/LAC mothers. Milk protein content was significantly related to weight gain. These data suggest that neonatal body weight gain can be accelerated in nursing infants whose mothers have received GH from at least the second trimester of pregnancy through the lactational interval. Since infants of mothers receiving GH during lactation only were not different from controls, the effect of GH in this treatment paradigm may be mammogenic rather than galactopoietic per se.

Properties of a cleaved two-chain form of recombinant human growth hormone.

Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.

Pubertal growth spurt in the female rhesus monkey: Relation to menarche and skeletal maturation.

Three-monthly measurements of tibia length, crown-rump length (CRL), and body wieght have been made on nine indoor-housed and six outdoor-housed female rhesus monkeys (Macaca mulatta) from ages 17 to 41 months. In the indoor-housed there were clear pubertal growth spurts, identifiable in individual curves, with average peaks at 22.5 months for tibia length, 23.5 months for CRL, and 24.5 for weight. Menarche occurred on average at 26.0 months. In both skeletal dimensions the amount of acceleration exceeded that which occurs in man. The relative timing of the spurts and of menarche was similar to that in man. The outdoor-housed monkeys showed a similar spurt in tibia length, although a less easily identifiable one in crown-rump length. The peak velocity of tibia length in these monkeys occurred on average at 30.5 months, with menarche at 32.5 months. We conclude that the pubertal growth spurt in female rhesus is very little different from that in man.

Effects of growth hormone on the tempo of sexual maturation in female rhesus monkeys.

The signal that initiates and maintains the developmental change in LHRH and, consequently, LH secretion in primates, thus regulating the tempo of puberty, is not known. Given the close association between reproductive development and bone maturation, we examined the hypothesis that GH was involved in developmental increases in LH release, specifically by augmenting the decrease in estradiol (E2) negative feedback inhibition of LH that characterizes late puberty in primates. Recombinant human GH (rhGH; 250 micrograms/kg) was given (sc) three times weekly to immature female rhesus monkeys to determine if developmental increases in basal serum LH would occur at an earlier age, and if menarche and first ovulation also would be advanced. The study groups included intact females receiving rhGH (INT + GH; n = 5), intact control animals (INT; n = 6), ovariectomized females receiving E2 plus rhGH (E2OVX + GH; n = 5), and E2-treated ovariectomized control monkeys (E2OVX; n = 4). The females were studied from 20 months of age until their serum LH levels increased (E2OVX groups) or until the occurrence of first ovulation (intact groups). After 12 months of rhGH treatment, the crown-rump lengths were significantly increased, regardless of ovarian status, an effect maintained in the intact females through 21 months of treatment. The mean age at the time of the initial rise in serum LH was advanced by rhGH treatment in intact females (29.6 +/- 0.4 vs. 31.3 +/- 0.3 months), but not E2OVX females. Subsequent maturational elevations in LH secretion were similar in the E2OVS + GH and E2OVX animals even after an incremental increase in E2. Ages at menarche were similar in the INT + GH and INT groups, whereas first ovulation was significantly advanced in three of five INT + GH females (31.5 +/- 0.7 months) compared to that in INT females (43.5 +/- 0.3 months). The remaining INT + GH females ovulated at an age (42.4 +/- 0.4 months) similar to that of INT females. Those females that ovulated by 32 months had higher skeletal maturity scores than the later ovulating INT females, with the other INT + GH females being intermediate. Furthermore, rhGH resulted in a significant increase in serum E2 levels within 12 h of injection, which remained elevated through 24 h. This effect of rhGH on ovarian E2 secretion did not occur until females had shown elevations in basal serum LH.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of a natural versus artificial environment on the tempo of maturation in female rhesus monkeys.

To identify factors that regulate the tempo of growth and puberty, the present study examined how the environment influenced the timing of menarche and first ovulation in rhesus monkeys and how these events were related to differential rates of growth. Spring-born females were raised from 12 months of age under natural outdoor conditions (OH; n = 6) or indoors (IH; n = 9) under a controlled photoperiod (12h of light, 12h of darkness) and temperature (20-23 C). Ages at the initial increases in serum bioactive LH levels (27.7 +/- 0.7 vs. 31.4 +/- 1.0 months), menarche (26.0 +/- 0.7 vs. 32.5 +/- 0.9 months), and first ovulation (33.9 +/- 1.4 vs. 43.5 +/- 0.3 months) were significantly advanced in IH compared to OH females. First ovulation for the OH females occurred exclusively in October and November of the fourth year, whereas the distribution of first ovulation of IH females was biomodal, with seven of nine occurring in November or December at 31.8 +/- 0.5 months, and two of nine ovulating in September or October at 41.2 +/- 0.5 months. Serum levels of PRL varied seasonally in OH females throughout development, with peaks in July and nadirs in October. A similar rhythm was observed for IH females during the first 12 months of indoor housing, after which point the period decreased from 11.9 +/- 0.5 to 9.3 +/- 0.6 months. Overall increments in body weight did not differ between groups. An acceleration of growth in both crown-rump and tibial lengths occurred just before menarche in both groups, and this occurred at about 26 months for IH and about 32 months for OH females. Skeletal maturity was significantly advanced at 27 months in IH females and at every chronological age thereafter. Serum concentrations of somatomedin-C and GH paralleled group differences in bone maturation. Both hormones were significantly elevated by 16-18 months of age in IH animals compared to OH females and remained so until 34-36 months of age. Although a distinct seasonal rhythm in both GH and somatomedin-C was evident in OH animals, no such pattern was observed in IH females. These data suggest that exposure to an outdoor environment moderates the tempo of both sexual and skeletal maturation. The acceleration in reproductive development in animals exposed to a constant environment was associated with an acceleration in bone maturation, suggesting that common factors may be responsible for the initiation of both events.

Equivalent potency and pharmacokinetics of recombinant human growth hormones with or without an N-terminal methionine.

Two forms of human GH (hGH) have been produced by recombinant DNA technology. One form has an amino acid sequence identical to that of the natural pituitary hormone (rhGH) and the other form has an additional N-terminal methionine (Met-hGH). The biological potencies of these 2 polypeptides have been compared in hypophysectomized rats in a multidose study measuring body weights and several long bone growth parameters. The pharmacokinetic profiles after iv and sc injection were determined in cynomolgus monkeys in a 4-period cross-over study. All of the measured parameters in all the studies indicated that there was no difference in the two forms of hGH. Measurements taken after 27 daily injections of rhGH or Met-hGH (30-500 micrograms/kg.day) indicated that femur length and width of the proliferative zone in the tibial epiphysis showed dose-related effects for both forms of hGH but no difference between them. The relative potency, based on body weight gain, was calculated using a parallel line bioassay. Weight gain after 8 daily injections in the 5-dose long bone growth study indicated a rhGH potency of 0.80 (95% confidence interval, 0.5-1.23) relative to Met-hGH. It was concluded that the presence of an N-terminal methionine on hGH has no effect on potency in this model. The pharmacokinetic parameters after iv administration were estimated by fitting serum concentration-time data to a 2-compartment model. Parameters after sc injection were computed by compartment-independent methods. Met-hGH and rhGH had very similar pharmacokinetic profiles after both routes of administration. Comparison of the pharmacokinetic parameters indicated that the clearance after iv administration (rhGH, 15 ml/min; Met-hGH, 13 ml/min) and the sc bioavailability (rhGH, 0.72 +/- 0.21; Met-hGH, 0.59 +/- 0.21) were not significantly different for the 2 forms of hGH. It was concluded that rhGH and Met-hGH have equivalent bioavailability and pharmacokinetics in cynomolgus monkeys.

The synthetic 32-46 fragment of human growth hormone increases insulin and glucagon levels in the conscious dog.

Human growth hormone (hGH, 22 K) has an acute insulinlike effect not observed with the 20 K variant form of hGH that lacks amino acids 32 to 46 (deletion peptide, hGH32-46). The possibility that hGH32-46 increases insulin secretion was examined by infusing hGH32-46 (1.6 nmol/kg/min) or saline in a crossover design study into each of four conscious 16-hour-fasted dogs for three hours (0 to 180 minutes) following a 40-minute control period. At 90 minutes, plasma glucose was raised to and maintained at 170 mg/dL by glucose infusion for three hours (until 270 minutes). After a lag period of 30 minutes hGH32-46 infusion caused glucagon to increase (P less than 0.05) by 67 +/- 20 pg/mL and insulin tended to rise by 8 +/- 3 microU/mL. Saline tended to cause glucagon and insulin to decline slightly (by 17 +/- 8 pg/mL and 6 +/- 2 microU/mL); hGH32-46 increased (P less than 0.05) tracer determined (3H-3-glucose) glucose production by 1.13 +/- 0.66 mg/kg/min while saline had no effect. Neither treatment changed plasma glucose (100 +/- 4 to 105 +/- 3 mg/dL with hGH32-46; 99 +/- 4 to 99 +/- 4 mg/dL with saline). Induction of hyperglycemia (168 +/- 2 mg/dL) caused glucagon concentrations to fall similarly to about 50 pg/mL with and without hGH32-46. Insulin rose in both protocols but to a greater extent (P less than 0.05) with hGH32-46 (+67 +/- 18 v +35 +/- 13 microU/mL at 180 minutes).(ABSTRACT TRUNCATED AT 250 WORDS)

A new bioassay for human growth hormone based on incorporation of labelled proline into skin.

An in-vitro bioassay for human growth hormone (hGH) has been developed, based on the dose-related enhancement of radioactivity in skin of hypophysectomized rats given daily s.c. injections of hGH and a single i.p. injection of labelled proline. The measured radioactivity, taken as the response, was shown to be independent of the site at which the skin was sampled. Valid dose-response relationships were obtained after as little as 3 days of treatment with GH. The assays by this method are shown to be more precise than the widely used weight-gain and tibial-width bioassays. Assays carried out gave an average index of precision of 0.036.

Autoradiographic comparison of growth factors: influence of growth hormone and somatomedin B on patterns of proline incorporation.

Patterns of incorporation of 14C proline have been compared by whole body autoradiography to study the action of growth factors, infused for 1 week to hypophysectomized rats by subcutaneously implanted minipumps. Human growth hormone (hGH) caused dose-related increases in the width of the tibial epiphyseal cartilage and obvious weight gain, whereas only at 640 micrograms/week did Somatomedin B have a barely significant effect on these two parameters. Samples of tissues showing significant proline uptake by autoradiography were burned for direct measurement of 14C content. Both hGH (80-160 micrograms/week) and SMB (160-640 micrograms/week) significantly enhanced proline uptake in skin, tibia, liver and kidney. Whereas the Somatomedin was less active than hGH on skin it showed a greater effect on bone, significantly exceeding the apparently maximal response to hGH. The autoradiographic technique can be used not only to compare different growth factors but also to study the growth process by use of appropriate tracers.

Transdermal uptake of a peptide hormone: inhibition by calcitonin eardrops of induced osteolysis in guinea-pig ossicles.

In view of a recent proposal that calcitonin injections may arrest the bony pathology of otosclerosis, we have tested the possibility of obtaining locally effective concentrations by giving salmon calcitonin in eardrops. Osteolysis of guinea-pig ossicles induced by injecting parathyroid hormone shortly before explantation was markedly inhibited by 3 days prior instillation of the calcitonin in an aqueous vehicle or in dimethyl sulphoxide, but not by a solution in propylene carbonate.