Dose Of GHRH Research Articles

Repetitive Stimulation of the Pituitary with Growth-Hormone-Releasing Hormone Alters the Proportion of 22 and 20 Kilodalton Human-Growth Hormone Released

Background/Aims. 20 Kilodalton-hGH (20 K-hGH) is the second most abundant pituitary GH variant after 22 K-hGH. In the steady state the proportion of 20 : 22 K-hGH appears constant; does this proportion change with repetitive somatotroph stimulation? Methods. Forty adult males were randomised to receive a GHRH(1–29)NH2 bolus (0.5 μg/kg (n = 20) or 1.0 μg/kg (n = 20)), preceded or followed by a saline bolus, 1 week apart. Four to six weeks later, 10 subjects received 0.5 μg/kg GHRH(1–29)NH2 at 0, 60, 120, and 180 minutes. Clearance rate of 22 and 20 K-hGH was measured in 10 subjects. Results. Total amount/proportion of 22 K-hGH/20 K-hGH secreted was similar for both GHRH(1–29)NH2 doses. Repetitive stimulation reduced the amount of 22 K-hGH released whereas the amount of 20 K-hGH did not change significantly leading to an increase in the proportion of 20 K-hGH (P = .05). Half-life of 20 and 22 K-hGH were not significantly different (P = .55). Conclusions. Repetitive stimulation of the somatotroph may alter the proportion of GH variant released.

Pre-VersusPostmenopausal Age, Estradiol, and Peptide-Secretagogue Type Determine Pulsatile Growth Hormone Secretion in Healthy Women: Studies Using Submaximal Agonist Drive and an Estrogen Clamp

GH-releasing peptide (GHRP), GHRH, and somatostatin are physiological regulators of pulsatile GH secretion. Age, independently of abdominal visceral fat (AVF) and basal (nonpulsatile) GH secretion, damps pulsatile GH secretion driven by physiological (rather than pharmacological) amounts of GHRP and GHRH in an experimentally controlled estradiol (E(2)) milieu. A prospectively randomized, double-blind parallel-cohort study was conducted at an academic medical center. Community-dwelling healthy premenopausal (PRE, age 24 +/- 0.8 yr, n = 20) and postmenopausal (POST, age 63 +/- 1.8 yr, n = 22) women participated in the study. Gonadal-axis down-regulation with leuprolide was followed by randomized addback of placebo or transdermal E(2) and separate-day iv bolus injections of a half-maximally stimulatory dose of GHRP-2 or GHRH (each 0.33 mug/kg). Three-way analysis of covariance included main factors age, E(2) status, and secretagogue type and covariates AVF and basal GH secretion. Submaximally stimulated pulsatile GH secretion was positively determined by PRE vs. POST age (P < 0.001), E(2) repletion vs. depletion (P = 0.001) and GHRP-2 vs. GHRH stimulation (P < 0.001), after adjustment for AVF and basal secretion. E(2) vs. placebo elevated fasting mean GH concentrations in both PRE and POST women (P = 0.006) but increased basal (nonpulsatile) GH secretion in PRE only (P = 0.002). PRE vs. POST age prolonged GHRH-driven GH secretory bursts by 36% (P = 0.006). PRE vs. POST age, E(2) availability, and physiological peptide drive are triple determinants of pulsatile GH secretion independently of abdominal visceral fat and nonpulsatile GH secretion in healthy women.

Preservation of GHRH and GH-releasing peptide-2 efficacy in young men with experimentally induced hypogonadism

Somatostatin (SS), GHRH, GH-releasing peptide (GHRP), and the sex-steroid milieu regulate GH secretion. To test whether GHRH and GHRP remain effective secretagogs in the face of short-term hypogonadism. Prospective, randomized double-blind. Healthy young men (n=24) received a GnRH agonist twice 3 weeks apart followed by placebo (n=13, Pl) or testosterone (n=11, testosterone) addback. were then given consecutive i.v. infusions of l-arginine (to restrain SS outflow) and a maximally effective dose of GHRH or GHRP-2 (to test corresponding secretagog pathways). GH secretion stimulated by l-arginine/GHRH and by l-arginine/GHRP-2 was unaffected by combined testosterone/estradiol (E(2)) depletion. The low testosterone/E(2) milieu decreased basal (nonpulsatile) GH secretion (P=0.038), without altering fasting pulsatile GH secretion or IGF1 or IGF-binding protein (IGFBP)-3 concentrations. IGFBP-1 (P<0.0001) and abdominal visceral fat (AVF, P=0.017) correlated negatively with fasting basal GH secretion. By contrast, IGF1 (P=0.0012) and IGFBP-3 (P=0.015) correlated positively with fasting pulsatile GH secretion. AVF (P=0.0024) was a negative determinant, and IGF1 a positive determinant (P=0.018), of GHRH-driven GH pulses. Responses to GHRP-2 were unrelated to any of these factors. l-arginine/GHRP-2 appears to be an especially robust stimulus of GH secretion, since efficacy is unmodified by profound short-term hypogonadism, a range of AVF estimates, and a spectrum of IGF1, IGFBP-1, and IGFBP-3 concentrations. Whether robustness also applies to chronic hypogonadism is not known.

Insulin-like growth factor-I feedback regulation of growth hormone and luteinizing hormone secretion in the pig: evidence for a pituitary site of action

Three experiments (EXP) were conducted to determine the role of insulin-like growth factor-I (IGF-I) in the control of growth hormone (GH) and LH secretion. In EXP I, prepuberal gilts, 65 ± 6 kg body weight and 140 days of age received intracerebroventricular (ICV) injections of saline (n = 4), 25 μg (n = 4) or 75 μg (n = 4) IGF-I and jugular blood samples were collected. In EXP II, anterior pituitary cells in culture collected from 150-day-old prepuberal gilts (n = 6) were challenged with 0.1, 10 or 1000 nM [Ala15]-h growth hormone-releasing hormone-(1-29)NH2 (GHRH), or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 1000 nM GHRH. Secreted GH was measured at 4 and 24 h after treatment. In EXP III, anterior pituitary cells in culture collected from 150-day-old barrows (n = 5) were challenged with 10, 100 or 1000 nM gonadotropin-releasing hormone (GnRH) or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 100 nM GnRH. Secreted LH was measured at 4 h after treatment. In EXP I, serum GH and LH concentrations were unaffected by ICV IGF-I treatment. In EXP II, relative to control all doses of GHRH increased (P < 0.01) GH secretion. Only 1, 10, 30 nM IGF-I enhanced (P < 0.02) basal GH secretion at 4 h, whereas by 24 h all doses except for 30 nM IGF-I suppressed (P < 0.02) basal GH secretion compared to control wells. All doses of IGF-I in combination with 1000 nM GHRH increased (P < 0.04) the GH response to GHRH compared to GHRH alone at 4 h, whereas by 24 h all doses of IGF-I suppressed (P < 0.04) the GH response to GHRH. In EXP III, all doses of IGF-I increased (P < 0.01) basal LH levels while the LH response to GnRH was unaffected by IGF-I (P > 0.1). In conclusion, under these experimental conditions the results suggest that the pituitary is the putative site for IGF-I modulation of GH and LH secretion. Further examination of the role of IGF-I on GH and LH secretion is needed to understand the inhibitory and stimulatory action of IGF-I on GH and LH secretion.

Ghrelin improves growth hormone responses to growth hormone-releasing hormone in a streptozotocin-diabetic model of delayed onset

GH secretion is markedly altered in diabetes mellitus (DM) in both rats and humans, albeit in opposite directions. In the rat, diabetes suppresses pulsatile GH secretion, especially high amplitude pulses, and decreases GH responses to secretagogue, depending inversely on severity of metabolic alteration. In the present study, we wanted to address the GH responses to GHRH and low doses of ghrelin in a streptozotocin (STZ) model of diabetes characterized by the delayed onset of the metabolic alterations. We have shown that the administration of high doses of STZ (90 mg/kg in 0.01 M solution of chloride-sodium, ip) to five-day-old pups (n5-STZ) can induce the appearance of a characteristic diabetic syndrome in adult age, the diabetic triad, with elevated plasma glucose levels: polyuria, polydipsia, hyperphagia, and reduced body weight gain. At the age of 3 months, in these n5-STZ male and female rats the GH responses to GHRH (1 microg/kg) and GHRH combined with ghrelin (1+3 microg/kg) had diminished both in punctual times and in the area under the curve (AUC). However, the combined administration of GHRH and ghrelin, being the more potent stimulus, elicited a synergistic GH response. Thus, male and female rats with delayed onset diabetes displayed an altered GH response to GHRH, although the combined administration of GHRH and ghrelin was able to restore the GH secretion with a synergistic effect.

Differential Regulation of GHRH-Receptor and GHS-Receptor Expression by Long-Term In Vitro Treatment of Ovine Pituitary Cells with GHRP-2 and GHRH

GH secretion is regulated by GHRH and somatostatin via actions on their specific receptors in pituitary somatotropes. Ghrelin and synthetic analogs, GHRPs, also stimulate GH release via GHS-receptors (GHS-R). To examine the long-term effect of GHRH and/or GHRP on somatotropes, primary cultured ovine somatotropes were treated with GHRH (10(-9) and 10(-8) M) and GHRP-2 (10(-8) and 10(-7) M) for up to 2 d. After treatment, culture medium was collected for GH assay, and total RNA was extracted for RT-PCR analysis. To evaluate cell cultures used in this report, somatotrope-enriched pituitary cells were challenged by 6 h GHRH and dexamethasone (DEX) treatment. As expected, GHRH significantly decreased, whereas DEX increased, the levels of GHRHR mRNA. Combined low doses of GHRH (10(-9) M) and GHRP-2 (10(-8) M) treatment for 24 h increased accumulated GH secretion, significantly more than that induced by high doses of GHRH (10(-8) M) and GHRP-2 (10(-7) M). While levels of GHRH-R mRNA increased, GHS-R mRNA levels were decreased by low doses of GHRH and GHRP-2 for 24 h. High doses of GHRH and/or GHRP-2 for 2 d did not increase GH secretion in the second day of treatment and reduced the level of GHRH-R mRNA. High doses of GHRP-2 treatment decreased the levels of both GHRH-R and GHS-R mRNA. Low doses of GHRH and/or GHRP-2 for 2 d increased the level of GHS-R mRNA without changing GHRH-R mRNA levels. Such treatment also increased ghrelin- (10(-9) M) or ghrelin/GHRH (10(-9) M)-induced GH secretion. These results suggest that low doses of GHRP-2 and GHRH prime somatotropes for stimulation by GHRH and ghrelin.

Sustained Growth Hormone (GH) and Insulin-Like Growth Factor I Responses to Prolonged High-Dose Twice-Daily GH-Releasing Hormone Stimulation in Middle-Aged and Older Men

Postulated mechanisms underlying the relative hyposomato-tropism of aging include reduced hypothalamic drive by GHRH. To test this notion, we administered 1 mg (n = 11) vs. 4 mg (n = 11) recombinant human GHRH-1,44-amide s.c. twice daily for 3 months in a double-blind, parallel-cohort design to 22 healthy men (ages, 53-68 yr). After 3 months, GHRH elevated: overnight GH concentrations from 0.71 +/- 0.19 to 1.74 +/- 0.39 microg/liter (P < 0.001; 1 mg) and from 0.80 +/- 0.15 to 5.12 +/- 0.40 microg/liter (P < 0.001; 4 mg) and IGF-I concentrations from 117 +/- 14 to 234 +/- 20 microg/liter (P = 0.007; 1 mg) and from 147 +/- 13 to 286 +/- 22 microg/liter (P < 0.001; 4 mg). Only the higher GHRH dose also increased total body water (tritium space; P = 0.024) and fat-free mass (dual-energy x-ray absorptiometry; P = 0.021), and reduced total abdominal adiposity (computed axial tomography scan; P = 0.042). Both supplementation schedules shortened the time required to walk 30 m and ascend four flights of stairs (P < 0.025 each). Lower extremity strength, aerobic capacity, and bone mineral density did not change. Local injection site reactions were common. We conclude that sc administration of a large dose of GHRH (4 mg) twice daily for 3 months elevates GH and IGF-I concentrations, increases total body water and fat-free mass, reduces total abdominal adiposity, and enhances certain performance measures in healthy aging men but causes local skin reactions.

Growth hormone and aging?

„The medicalization of old age is slowly entering the endocrine clinic and life-style medication for the affluent adult is about to enter the mainstream of endocrinology“. Intriguing new findings: Single gene mutations have been identified that lead to increased longevity. A highly conserved regulatory system for aging exists. Increased longevity of hypopituitary dwarf mice and GH resistant knockout mice appears to be in contrast with observations made in clinical practice. In humans, patients with a PROP-1 gene mutation can survive to a very advanced age, apparently longer than normal individuals in the same population. These findings may challenge the importance of GH as antiaging drug, and the notion that GH deficiency may shorten life span. On the other hand, reduced longevity in untreated patients with congenital isolated GH deficiency (GH-1 gene deletion) suggests that the lack of GH might have an impact on aging. Four retrospective studies and one prospective have all confirmed increased mortality in patients with hypopituitarism compared with age-matched controls, but the exact cause remains unclear due to the existence of limiting factors. There are no data whether or not GH replacement will prolong life expectancy. Somatopause and GH deficiency in elderly: Against a background of declining GH secretion and serum IGF-I with aging, clinical syndrome of GHD translates into profound changes in body composition and function. The GH Research Society consensus statement on the diagnosis of GHD, however, states that in adults, hypothalamic-pituitary disease must be present before the diagnosis of GHD can be considered. GH secretion in the patients with hypothalamo-pituitary disease is only 12%of that observed in the controls. Thus these patients can be distinguished from hyposomatotropism of ageing. Some studies (but not all) have reported rather convincing positive effects of GH replacement. GH replacement in elderly patients with organic GHD should be included in clinical consideration particularly when designing the individual replacement therapy. Changes in the functional activity of GHRH and somatostatin synthesizing neurons in the hypothalamus play a role in the age-related GH hyposecretion. In the last 10 years the unnatural growth hormone secretagogues (GHS) with the isolation of the endogenous ligand for the GHS-receptor, ghrelin, demonstrate the existence of a third physiologic system of GH regulation and raise the issue of the GHS/ghrelin function in aging. Very old subjects after combined, acute administration of saturating doses of GHRH and GHS, show GH response to be age-independent and fully preserved. Recently orally active GHS in healthy older men showed sustained GH increase for 2 yr. Overall these findings suggest that in aged individuals GH secretion may be restored by pharmacological means, based on the ability of GHSs to correct the attenuated GH pulsatility. The notion of „disease“ is a slippery one and health is equally impossible to define: Recent research efforts have focused on identifying mediators of debilitating processes or markers of poor health. Clinical studies linking catabolic cytokines, circulating levels of IGF-I, sarcopenia with mortality in very old community-dwelling men and women has shown that greater production of catabolic cytokines and reduced IGF-I levels are associated with increased mortality. In the Rotterdam study an association between a polymorphism of the IGF-I gene and total IGF-I serum levels, birthweight, body height and the risk for developing diabetes and cardiovascular diseases later on in life, has been found. Only homozygous carriers of the polymorphism of the IGF-I gene had an age-related and GH-dependent decline in circulating total IGF-I levels. Further research efforts should focus on identifying groups of patients in whom the risk of adverse effects is outweighed by the benefits achieved from GH treatment particularly in the elderly.

GH responses to GHRH and GHRP-6 in Streptozotocin (STZ)-diabetic rats

GH responses to GHRH, the physiologic hypothalamic stimulus, and GHRP-6, a synthetic hexapeptide that binds the Ghrelin receptor, were studied in rats treated with streptozotocin (STZ), an experimental model of diabetes. Sprague–Dawley male rats received a single injection either of STZ (70 mg/Kg in 0.01 M SSC, i.p.) or of the vehicle (0.01 M SSC). GH responses were challenged with two different doses of GHRH (1 and 10 μg/kg) or GHRP-6 (3 and 30 μg/kg) and with a combination of both at low (1 + 3 μg/kg) or high (10 + 30 μg/kg) doses, respectively. We observed a dose-dependent effect for GH responses to GHRH both in STZ-treated rats and in controls. However, we could not find significant differences between STZ-rats and controls. GH responses to GHRP-6 occurred in a dose-dependent manner in STZ-rats, but not in controls. GH responses to GHRP-6 in both groups were clearly lower than those elicited by GHRH. GH responses to 30 μg/Kg of GHRP-6 were significantly greater in STZ-rats than in controls (AUC: 3549.9 ± 1001.4 vs. 2046.4 ± 711.7; p < 0.05). The combined administration of GHRH plus GHRP-6 was the most potent stimuli for GH in both groups. The administration of doses in the lower range (1 + 3 μg/Kg, GHRH + GHRP-6 respectively) induced a great peak of GH in STZ-rats and in control rats, revealing a synergistic effect of GHRH and GHRP-6 in both groups. When the higher doses were administered (10 + 30 μg/kg), GH levels in time 5, and AUC were significantly higher in control rats. In addition, a negative correlation between WT (weight tendency) values and GH responses, represented as AUC, could be established in STZ-rats (r 2 = −0.566, p = 0.004 for GHRH; r 2 = −0.412, p = 0.028 for GHRP-6). Thus, the more negative the values of WT were, the more severe the metabolic alteration and, therefore, the higher the GH response to GHRH and GHRHP-6. In conclusion, our results do not support the existence of a functional hypothalamic hypertone of SS in diabetic rats, as GH responses were not usually reduced in STZ-rats, except when both secretagogues were administered together at the higher doses. Besides, GH responses to GHRH and GHRP-6 were inversely correlated with the severity of the metabolic alteration in STZ-rats, meaning that worse glycaemic control promoted higher GH secretion. These results resemble those found in humans, where GH responses to secretagogues are increased in type-1 diabetes and depend on hyperglycaemia, and are representative of not well-controlled insulin-dependent diabetic status.

Use of growth hormone response to growth hormone-releasing hormone to determine growth potential in beef heifers.

The response of GH to GHRH at weaning is known to predict postweaning growth and body composition in beef bulls. The objective of this study was to determine whether GH response to a challenge of GHRH and plasma IGF-I can predict growth rate and body composition in the beef heifer. Growth hormone response to a challenge with two doses of GHRH was measured in 67 Angus heifers averaging 225 d of age (SD = 21) and 217 kg BW (SD = 32). Blood samples were collected at 0 and 10 min relative to an initial "clearance dose" (4.5 micrograms GHRH/100 kg BW) and again, 3 h later, relative to a challenge dose (1.5 or 4.5 micrograms GHRH/100 kg BW). Each animal received each of the two challenge doses, which were randomly assigned across 2 d of blood collection. Serum GH concentration was measured by RIA. Plasma was collected every 28 d during a 140-d growth test and assayed for IGF-I by RIA. Body weight was measured every 28 d and hip height was measured at weaning and at the end of a 140-d growth test. Average daily gain was calculated on d 140 of the growth test and body composition measurements were estimated by ultrasound 2 wk after completion of the growth test. Responses to the two GHRH challenges were dose-dependent (P < 0.05). Average daily gain tended to be related to GH response to the 1.5 micrograms GHRH/100 kg BW dose (R2 = 0.05; P = 0.06), but no relationship was observed at the 4.5 micrograms GHRH/100 kg BW dose (R2 = 0.00; P = 0.93). An inverse relationship (R2 = 0.06; P = 0.02) was observed between response to the 1.5 micrograms GHRH/100 kg BW dose and intramuscular fat percentage. Mean plasma IGF-I concentration was positively associated with ADG (R2 = 0.06; P < 0.01). Growth hormone response to GHRH is modestly related to body composition but not to ADG in weanling beef heifers and likely has limited use in evaluation of growth performance in replacement beef heifers.

Growth hormone response to growth hormone-releasing hormone in beef cows divergently selected for milk production.

In dairy cattle, increased circulating growth hormone has been associated with selection for greater milk yield. This study tested the hypothesis that beef cows divergently selected for milk production would have differing GH responses to a challenge dose of GHRH. Growth hormone response to a challenge of GHRH was measured in 36 Angus-sired cows ranging from 6 to 10 yr of age. The cows were classified as high milking (n = 16) or low milking (n = 20), on the basis of their sires' milk EPD. Mean milk EPD (in kilograms) were 16.6 and -14.4 for high and low milking cows, respectively. Milk production was estimated by the weigh-suckle-weigh procedure. Blood samples were taken immediately before and 10 min after a clearance dose of 4.5 microg of GHRH/100 kg BW (injected i.v.) and, 3 h later, immediately before and 10 min after a challenge dose of either 1.5 or 4.5 microg of GHRH/100 kg BW. Each animal received both challenge doses, and the doses were randomly assigned across 2 d of blood collection. Serum concentrations of GH and IGF-I were measured by RIA. Serum IGF-I was measured in the baseline blood sample on d 1 of blood collection. A positive relationship (r = 0.35; P = 0.03) was observed between the cows' rankings for each dose of GHRH; that is, high responders to the low dose were high responders to the high dose. Growth hormone response to the 4.5 microg/100 kg BW challenge dose of GHRH was positively related to sire milk EPD (R2 = 0.09; P = 0.03). Response of GH to the 1.5 microg GHRH/100 kg BW challenge dose also tended to be related (P = 0.08) to sire milk EPD of high milking cows. In addition, IGF-I concentrations of high milking cows were inversely related (R2 = 0.24; P = 0.04) to sire milk EPD. Growth hormone response to GHRH challenge may have potential as an additional tool in the evaluation of milk production in beef cattle.

Ghrelin elicits a marked stimulatory effect on GH secretion in freely-moving rats.

Ghrelin is a growth hormone-releasing acylated peptide from stomach. The purified peptide consist of 28 amino acids in which the serine 3 residue is n-octanoylated. Ghrelin has been reported to increase in vitro GH secretion as well as in vivo plasma GH levels in pentobarbital anaesthetized rats. The aim of this work was to characterize the stimulatory effect of Ghrelin on in vivo GH secretion in freely-moving rats. Furthermore, we compare the effect of Ghrelin with GHRH. In addition to vehicle, we administered different doses of Ghrelin (3 nmol/Kg, 12 nmol/Kg and 60 nmol/Kg); GHRH (3 nmol/Kg and 12 nmol/kg). Plasma GH levels were measured in blood samples taken at 5, 10, 15, 20, 30 and 45 min after their administration as an i.v. bolus at 0 min. Administration of Ghrelin led to an increase in plasma GH levels at all time-points tested (5, 10, 15, 20 and 30 min, P<0.01; and 45 min, P<0.05) in comparison to control untreated rats. A maximal stimulatory effect on plasma GH was observed following administration of 12 nmol/Kg of Ghrelin, the effect being similar to the one obtained with 60 nmol/Kg in terms of both AUC and mean peak GH levels. At the dose of 3 nmol/Kg GHRH and Ghrelin exhibited a similar stimulatory effect in term of both, AUC and mean peak GH levels. However following administration of a dose of 12 nmol/Kg, the effect of Ghrelin was much greater than the same dose of GHRH in terms of both AUC and mean peak GH levels. In summary, this study provides the first evidences that Ghrelin exert a marked stimulatory effect in plasma GH levels in freely-moving rats and provides further evidences that Ghrelin may play an important role in the physiological control of GH secretion.

Leptin increases in vivo GH responses to GHRH and GH-releasing peptide-6 in food-deprived rats.

Leptin has recently been shown to have a stimulatory effect on basal GH secretion. However, the mechanisms by which leptin exert this effect are not yet clear. GHRH and GH-releasing peptide (GHRP)-6 are the two most potent GH secretagogues described to date. To determine if leptin could also enhance in vivo GH responses to a maximal dose of GHRH. Leptin (10microg i.c.v.) or vehicle was administered at random before GHRH (10microg/kg i,v.) or GHRP-6 (50microg/kg i.v.), to freely-moving rats with food available ad libitum and to (48h) food-deprived rats. Leptin and GH concentrations were measured by radioimmunoassay. Comparison between the different groups was assessed by the Mann-Whitney test. In comparison with fed rats, food-deprived rats showed a marked decrease in GH responses to GHRH as assessed by the area under the curve (5492+/-190ng/ml in fed rats and 1940+/-128ng/ml in fasted rats; P<0.05) and GHRP-6 (3695+/-450 in fed rats and 1432+/-229 in fasted rats; P<0.05). In comparison with its effects in vehicle-treated rats, leptin administered to food-deprived rats markedly increased GH responses to both GHRH (6625+/-613ng/ml; P<0.05) and GHRP-6 (5862+/-441ng/ml; P<0.05). These data suggest that the blunted GH response to GHRH and GHRP-6 in food-deprived rats is a functional and reversible state, and that the decreased leptin concentrations could be the primary defect responsible for the altered GH secretion in food-deprived rats.

Ipamorelin, a new growth-hormone-releasing peptide, induces longitudinal bone growth in rats.

Ipamorelin is a new and potent synthetic pentapeptide which has distinct and specific growth hormone (GH)-releasing properties. With the objective of investigating the effects on longitudinal bone growth rate (LGR), body weight (BW), and GH release, ipamorelin in different doses (0, 18, 90 and 450 microg/day) was injected s.c. three times daily for 15 days to adult female rats. After intravital tetracycline labelling on days 0, 6, and 13, LGR was determined by measuring the distance between the respective fluorescent bands in the proximal tibia metaphysis. Ipamorelin dose-dependently increased LGR from 42 microm/day in the vehicle group to 44, 50, and 52 microm/day in the treatment groups (P<0.0001). There was also a pronounced and dose-dependent effect on BW gain. The treatment did not affect total IGF-I levels, IGFBPs, or serum markers of bone formation and resorption. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the metaphysis of the tibia did not change significantly with treatment. The responsiveness of the pituitary to a provocative i.v. dose of ipamorelin or GHRH showed that the plasma GH response was marginally reduced (P<0.03) after ipamorelin, but unchanged after GHRH. The pituitary GH content was unchanged by ipamorelin treatment. Whether ipamorelin or other GH secretagogues may have a place in the treatment of children with growth retardation requires demonstration in future clinical studies.

Predicting bull growth performance and carcass composition from growth hormone response to growth hormone-releasing hormone.

Development of practical, physiologically based methods that provide an early, yet accurate, evaluation of a bull's genetic merit could benefit the beef industry. The use of GH response to a single, acute dose of GHRH was evaluated as a predictor of future growth performance and carcass characteristics of weanling bulls. Fifty-six Angus bulls averaging 229 d (SD = 27) of age were administered three doses i.v. (0, 1.5, and 4.5 microg/100 kg BW) of human GHRH (1-29) analog in a Latin square design balanced for residual effects. Blood samples were collected via jugular catheter at -60, -45, -30, -15, 0, 5, 10, 15, 30, 45, 60, 90 and 120 min relative to GHRH injection. Serum concentrations of GH were plotted over time. Response to GHRH was calculated as the area under the GH response curve (AUC-GH) using the trapezoidal approximation. Relationships between AUC-GH, weaning weight adjusted to 205 d of age (205-d WW), and direct weaning weight EPD (WWEPD) versus age-adjusted BW (BWadj), ADG, and carcass measurements from a 140-d growth performance test were evaluated using simple linear regression. A positive correlation between AUC-GH and ADG and an inverse relationship between AUC-GH and carcass fat were observed. The present study provides evidence that AUC-GH is a better predictor of future growth performance in beef bulls than 205-d WW or WWEPD values. Thus, GH response to GHRH is associated with subsequent growth and may be a useful tool for sire selection in beef production.

Age-related variations in the neuroendocrine control, more than impaired receptor sensitivity, cause the reduction in the GH-releasing activity of GHRPs in human aging.

The mechanisms underlying the reduction in the GH-releasing activity of GHRPs in aging are still unclear. Aim of our study was to verify in man whether age-related impairment of the neurohormonal control of GH secretion and/or receptor alterations are involved in the reduced GH response to GHRPs in aging. To this goal, in 16 normal elderly subjects (E, 66-81 yr) and 12 young controls (Y, 24-28 yr) we studied the effects of 1.0, 2.0 and 3.0 micrograms/kg i.v. Hexarelin (HEX), a synthetic hexapeptide, or GHRH, as well as the interaction among HEX (2.0 micrograms/kg), GHRH (2.0 micrograms/kg) and arginine (ARG, 0.5 gr/kg) on GH secretion. In Y the GH response to increasing doses of HEX (1.0 vs. 2.0 vs. 3.0 micrograms/kg; AUC0;v-120 +/- SEM: 1728.4 +/- 406.4 vs. 2265.9 +/- 298.4 vs. 2934.3 +/- 482.2 micrograms/L/h, p < 0.05 for 1.0 vs. 2.0 micrograms/kg) and GHRH (649.6 +/- 111.4 vs. 792.2 +/- 117.6 vs. 1402.6 +/- 363.0 micrograms/L/h) showed a progressive increase. Two micrograms/kg HEX and 1 microgram/kg GHRH were the maximal effective doses. Similarly, in E the GH response to increasing doses of HEX (336.7 +/- 50.0 vs. 742.8 +/- 157.9 vs. 1205.1 +/- 178.1 micrograms/L/h, p < 0.05 for 1.0 vs. 2 micrograms/kg, p < 0.001 for 1.0 vs. 3.0 micrograms/kg and p < 0.03 for 2.0 vs. 3.0 micrograms/kg) and GHRH (183.8 +/- 27.3 vs. 260.9 +/- 17.3 vs. 356.1 +/- 46.3 micrograms/L/h, p < 0.005 for 1.0 vs. 3.0 micrograms/kg and p < 0.05 for 2.0 vs. 3.0 micrograms/kg) showed a progressive increase. In E the GH response to 3 micrograms/kg HEX or GHRH were clearly higher than those to 2 micrograms/kg. However, at each dose the GH responses to HEX or GHRH in E were lower (p < 0.05) than those in Y. In Y the GH response to HEX + GHRH was synergistical (4259.2 +/- 308.0 micrograms/L/h, p < 0.05). ARG strikingly potentiated the GHRH-induced GH rise (2640.8 +/- 273.6 micrograms/L/h, p < 0.01) but not the HEX-induced one (2371.7 +/- 387.2 micrograms/L/h) as well as the synergistical effect of HEX and GHRH (4009.1 +/- 360.8 micrograms/L/h). In E the GH response to HEX and GHRH was still synergistical (1947.7 +/- 306.0 micrograms/L/h, p < 0.05) but these responses were lower than those in young (p < 0.01). On the other hand, in E ARG restored the GH response to GHRH (1858.9 +/- 172.8 micrograms/L/h, p < 0.01) and even those to HEX (2069.5 +/- 528.7 micrograms/L/h, p < 0.01) and HEX + GHRH (4406.0 +/- 1079.2 micrograms/L/h, p < 0.05). Our present results indicate that the impairment of GHRP and GHRH receptor activity may have a role in the reduction of the somatotrope responsiveness in aging. However, the age-related reduction in the GH-releasing activity of GHRPs seems mainly dependent on age-related variations in the neural control, i.e. concomitant GHRH hypoactivity and somatostatinergic hyperactivity.

Hypothalamo-pituitary-IGF-1 axis in female rats made obese by overfeeding

A gender-related impairment of the somatotrophic axis is present in obese Zucker rats, female rats being better preserved than males. We showed that another animal model of obesity, i.e., male rats made obese by feeding a hypercaloric diet had a reduced function of somatotrophic axis which was likely related to impairment of gonadal function. Aim of this work was that of studying the function of somatotrophic axis in female overfed rats and comparing it to that of male rats of the previous study. Sprague-Dawley female rats were fed an energy-rich palatable diet for seven months. At the end of overfeeding, according to the degree of overweight, rats were divided into overtly obese (Obese), overweight (Overweight) and Non-Obese, i.e. rats whose weights were similar to those of controls. Rats fed ad libitum with the standard pellet chow served as controls (Controls). Acute administration of a supramaximal dose of GHRH (2μg/rat, iv) elicited a plasma GH rise similar to that of Controls in all the groups, except in Obese which had a lower GH response. Growth hormone responses after GHRH administration were inversely related to plasma levels of free fatty acids (FFA). Pituitary GH content and gene expression as well as hypothalamic GHRH and SS mRNA content, were similar in all experimental groups and in Controls and the same was true for plasma concentrations of free IGF-I. These results indicate that, similarly to obese female Zucker rats, also overfed female rats had a better preservation of the somatotrophic axis than their male counterparts. In diet-induced obese rats, also the etiology of the impairment of somatotrophic axis seems to be gender-related i.e. due to a reduction of gonadal function in males and to an elevation of FFA in females.

Enrichment of type I and type II rat somatotrophs: characterization of growth hormone secretion in vitro.

Enriched fractions of heavily granulated (type II) and sparsely granulated (type I) somatotrophs have been prepared from male Sprague-Dawley rats by Percoll density gradient centrifugation in two steps. After 3 days of culture, basal GH release was 0.116 +/- 0.024 (n = 30) and 0.223 +/- 0.034 ng GH/microgram protein-min (n = 34) in type I and type II cells, respectively (P < 0.05). GH-releasing hormone (GHRH; 0.01-10 nM) stimulated GH release in type II cells, whereas type I cells only responded to higher doses of GHRH (1 and 10 nM). The dynamics of GH release were similar in the two cell types. Type II cells released more GH in absolute values, which may reflect the higher GH content in these cells. The somatostatin analog octreotide (100 pM) reduced basal GH release by 63% in type I cells, but by only 17% in type II cells. Octreotide also had a slightly greater effect on GHRH-induced GH release in type I cells. Both cell types responded to 100 nM GH-releasing peptide-6. We conclude that both type I and type II somatotrophs contribute to GH release, but type II cells are more sensitive to and release more GH when stimulated with GHRH. The role of type I cells may be to boost the initial secretory response at the onset of physiological pulses.

Growth hormone response to growth hormone-releasing hormone varies with the hypothalamic-pituitary abnormalities

We determined growth hormone (GH) and insulin-like growth factor I (IGF-I) levels after a 3 h infusion of escalating doses of growth hormone-releasing hormone (GHRH(1-29)) followed by a bolus injection in hypopituitary patients with marked differences in pituitary features at magnetic resonance imaging (MRI) in order to evaluate further the contribution of MRI in the definition of pituitary GH reserve in GH-deficient patients. Twenty-nine patients (mean age 14.5 +/- 4.0 years) were studied. Group I comprised 13 patients: seven with isolated GH deficiency (IGHD) (group Ia) and six with multiple pituitary hormone deficiency (MPHD) (group Ib) who had anterior pituitary hypoplasia, unidentified pituitary stalk and ectopic posterior pituitary at MRI, Group II consisted of eight patients with IGHD and small anterior pituitary/empty sella, while in group III eight had IGHD and normal morphology of the pituitary gland. Growth hormone and IGF-I levels were measured during saline infusion at 08.30-09.00 h, as well as after infusion of GHRH (1-29) at escalating doses for 3h: 0.2 micrograms/kg at 09.00-10.00 h, 0.4 micrograms/kg at 10.00-11.00 h, 0.6 micrograms/kg at 11.00-12.00 h and an intravenous bolus of 2 micrograms/ kg at 12.00 h. In the group I patients, the peak GH response to GHRH(1-29) was delayed (135-180 min) and extremely low (median 2mU/l). In group II it was delayed (135-180 min), high (median 34.8 mU/l) and persistent (median 37.4 mU/l at 185-210 min). In group III the peak response was high (median 30.8 mU/l) and relatively early (75-120 min) but it declined rapidly (median 14.4 mU/l at 185-210 min). In one group I patient, GH response increased to 34.6 mU/l. The mean basal value of IGF-I levels was significantly lower in group I (0.23 +/- 0.05 U/ml) than in groups II (0.39 +/- 0.13U/ ml, p < 0.01) and III (1.54 +/- 0.46 U/ml, p < 0.001) and did not vary significantly during the GHRH(1-29) infusion. The present study demonstrates that the impaired GH response to 3 h of continuous infusion of escalating doses of GHRH(1-29) was strikingly indicative for pituitary stalk abnormality, strengthening the case for use of GHRH in the differential diagnosis of GH deficiency. The low GH response, more severe in MPHD patients, might be dependent on the residual somatotrope cells, while the better response (34.6 mU/l) in the group Ia patients might suggest that prolonged GHRH infusion could help in evaluating the amount of residual GH pituitary tissue. Pituitary GH reserve, given the GH response to GHRH infusion in GH-deficient patients with small anterior pituitary/empty sella, seems to be maintained.

In vitro pituitary GH secretion after GHRH, forskolin, dibutyryl cyclic-adenosine 3′,5′-monophosphate and phorbol 12-myristate 13-acetate stimulation in long-term orchidectomized rats

In the present work in vitro GH pituitary responsiveness to GHRH in short-term (STO) and long-term orchidectomized (LTO) male rats was compared. In agreement with previous data obtained in vivo, pituitaries from STO rats showed reduced GH release after GHRH stimulation while LTO male pituitaries presented responses similar to those from control animals after maximal GHRH (10(-6)M) stimulation. This suggests that compensatory mechanisms have taken place, probably at the pituitary level, in order to restore GH pituitary responsiveness to high doses of GHRH. However, LTO male rats showed a reduced sensitivity to GHRH relative to intact males, as indicated by a higher EC50 vs controls (40.82 +/- 12.03 nM vs 0.35 +/- 0.09 nM in intact males). We aimed to investigate further the events involved in the compensatory mechanisms that take place in LTO rats. For this purpose, we compared in vitro GH secretion by pituitaries from intact and LTO male rats after stimulation with specific activators of the signal transduction pathways related to GH release. Forskolin and dibutyryl cyclic-adenosine 3',5'-monophosphate were more effective in eliciting GH secretion (expressed in terms of percent increment over basal GH release) in LTO males, whereas phorbol 12-myristate 13-acetate was completely ineffective in stimulating GH release in this group. Thus, our results clearly showed that long-term orchidectomy enhances the effectiveness of the cAMP pathway in inducing GH release while it completely blunts that of the protein kinase C pathway. In conclusion, orchidectomy decreased the effectiveness of GHRH in eliciting GH release in vitro. However, long-term orchidectomy activated compensatory mechanisms that restored complete GH pituitary responsiveness to maximal GHRH stimulation. These mechanisms seem not to operate in STO rats. An increased effectiveness of the cAMP pathway in eliciting GH release in LTO rats is probably involved in the aforementioned compensatory mechanisms.