Safety, tolerability, pharmacokinetics and pharmacodynamics of AZP-531, a first-in-class analogue of unacylated ghrelin, in healthy and overweight/obese subjects and subjects with type 2 diabetes.

AIM: To explore the safety, pharmacokinetics and pharmacodynamics in humans of the unacylated ghrelin analogue AZP-531, designed to improve glycaemic control and reduce weight. METHODS: Assessments, including glucose measurements, were performed in a three-part randomized study. In Part A, healthy subjects [n = 44, age 18-50 years, body mass index (BMI) 20-28 kg/m(2) ] received a single subcutaneous dose of 0.3, 3, 15, 30, 60 or 120 µg/kg AZP-531 or placebo. In Part B, overweight/obese subjects (n = 32, age 18-65 years, BMI 28-38 kg/m(2) ) and in Part C, patients with type 2 diabetes [T2D; n = 36, age 18-65 years, BMI 20-40 kg/m(2) , glycated haemoglobin (HbA1c) 7-10%] received AZP-531 or placebo for 14 days (daily doses of 3, 15, 30 or 60 µg/kg and 15, 2 × 30 or 60 µg/kg, respectively). RESULTS: AZP-531 was well tolerated. Single- and multiple-dose pharmokinetic variables were similar. Maximum AZP-531 concentrations were typically reached at 1 h post-dose. Observed maximum concentration (Cmax ) and area under the curve were dose-proportional. The mean terminal half-life (t1/2 ) was 2-3 h. In Part B, AZP-531 doses of ≥15 µg/kg significantly improved glucose concentrations, without increasing insulin levels, suggesting an insulin-sensitizing effect. AZP-531 decreased mean body weight by 2.6 kg (vs 0.8 kg for placebo). In Part C, glucose variables improved in all groups, including placebo, suggesting a study effect in uncontrolled patients at baseline. Notwithstanding, AZP-531 60 µg/kg reduced HbA1c by 0.4% (vs 0.2% for placebo) and body weight by 2.1 kg (vs 1.3 kg for placebo). CONCLUSIONS: AZP-531 was well tolerated in this first-in-human study. Its pharmacokinetic profile, suitable for once-daily dosing, and metabolic effects support further clinical development for T2D.

The continuous infusion of acylated ghrelin enhances growth hormone secretion and worsens glucose metabolism in humans.

CONTEXT: Acylated ghrelin (AG) has been discovered as a natural ligand of the GH secretagogue receptor type 1a and is now recognized as an important orexigenic factor. Besides stimulation of GH secretion and appetite, it exerts other central and peripheral actions including modulation of insulin secretion, glucose and lipid metabolism. OBJECTIVE: To define the effects of the continuous iv infusion of AG in humans with particular attention to metabolic parameters. MATERIALS AND METHODS: We studied the effects of 16- h (from 21:00 to 13:00 h) infusion of AG (0.5 microg/kg/h) or saline in 8 young volunteers who were provided with isocaloric balanced meals. GH, cortisol, insulin, glucose, free fatty acid (FFA), and ghrelin levels were assayed every 20 min. RESULTS: AG infusion increased circulating total ghrelin to a steady state that was maintained over 16 h infusion of the peptide. With respect to saline, AG infusion significantly modified GH, cortisol, insulin, and glucose profiles and decreased FFA area under the curve (p<0.01). AG increased GH pulse frequency and approximate entropy (p<0.05). AG enhanced the glucose response to both dinner (p<0.02) and breakfast (p<0.03). AG infusion blunted the early insulin response to dinner (p<0.03) but enhanced the second-phase insulin response to dinner and breakfast (p<0.05). CONCLUSIONS: The continuous exposure to AG in humans enhances somatotroph secretion but also worsens glucose metabolism, although it inhibits lipolysis. These findings in normal young volunteers are consistent with data from studies in animals and suggest that acylated ghrelin is likely to play a negative role in glucose metabolism.

Serum insulin-like growth factor binding protein-3 in the hypophosphatemic mouse: decreased activity and abnormal modulation by dietary phosphate.

The hypophosphatemic mouse, the murine homologue of X-linked hypophosphatemia, is characterized by renal defects in phosphate reabsorption and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) production and by an osteoblast dysfunction. In view of the potential importance of insulin-like growth factors (IGFs) in the regulation of these processes and the role of IGF-binding proteins (IGFBPs) as modulators of IGF action, we asked whether Hyp mice have alterations in IGFs or IGFBPs. Using specific radioimmunoassays and Western ligand blot analysis, we evaluated serum levels of IGFs (IGF-1 and IGF-II) and IGFBPs, respectively, in normal and Hyp mice. We also examined the effect of dietary phosphatase on these parameters. Serum levels of IGF-1 and IGF-II in Hyp mice were not significantly different from those in normal mice, but IGFBP-3 levels were significantly lower (70% of normal, p < 0.05) in the mutant strain. The other IGFBP species appear unchanged. Phosphate supplementation normalized serum phosphate levels in Hyp mice and elicited a significant decrease in serum IGF-I levels (23%, p < 0.05) and a further deduction in IGFBP-3 (22%, p < 0.02). Phosphate deprivation induced hypophosphatemia IGF-II. The present results indicate that the low serum IGFBP-3 activity in Hyp mice is not related to hypophosphatemia per se. Based on the documented effects of parathyroid hormone (PTH) on IGF-I and IGFBP-3, we propose that the secondary hyperparathyroidism displayed by Hyp mice and its exacerbation by phosphate supplementation may contribute to low IGFBP-3 levels in control Hyp mice and to the decreases in serum IGF-I and IGFBP-3 in phosphate-supplemented Hyp mice.

Alterations of pituitary growth hormone-releasing factor binding sites in aging rats.

This study was designed to determine whether growth hormone (GH)-releasing factor (GRF) binding sites are altered in parallel to the diminution of GH secretion that occurs in aging. Using [125I-Tyr10] hGRF (1-44)NH2 as radioligand, we performed cold saturation studies in 2, 8, 14 and 18-month-old Sprague-Dawley male rat pituitary homogenates. In young rats (2 months), analysis by Ligand revealed the presence of two distinct classes of binding sites (KDs = 0.86 +/- 0.15 and 400 +/- 210 nM; BMAXS = 269 +/- 47 fmol and 42 +/- 19 pmol/mg protein, respectively). In 8 and 14-month old rats, there was a concomitant decrease in capacity of the high affinity class of sites (P less than 0.01) and increase in capacity of the low affinity class of sites (P less than 0.05). In parallel, a transient increase in affinity of the high affinity class of sites was observed in 14-month-old rats (P less than 0.05). In old rats (18 months), the data were no longer statistically analyzed with a two site-model, indicating a severe blunting of the high affinity sites. As the GRF-induced GH secretion is still not diminished at 8 month of age in these animals, our results indicate: 1) that alterations of GRF binding sites precede the GH impairment, thus probably participate in the initiating of this phenomenon and 2) that the biological actions of GRF on GH secretion are likely mediated by the high affinity class of sites.

Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans.

Ghrelin possesses strong GH-releasing activity but also other endocrine activities including stimulation of PRL and ACTH secretion, modulation of insulin secretion and glucose metabolism. It is assumed that the GH secretagogue (GHS) receptor (GHS-R) 1a mediates ghrelin actins provided its acylation in Serine 3; in fact, acylated ghrelin only is able to exert endocrine activities. Acylated ghrelin (AG) is present in serum at a 2.5 fold lower concentration than unacylated ghrelin (UAG). UAG, however, is not biologically inactive; it shares with AG some non-endocrine actions like cardiovascular effects, modulation of cell proliferation and even some influence on adipogenesis. Thus, these actions are likely to be mediated by GHS-R subtypes able to bind ghrelin independently of its acylation. In order to further clarify whether UAG is really devoid of any endocrine action, we studied the interaction of the combined administration of AG and UAG (1.0 microg/kg i.v.) in 6 normal young volunteers (age [mean +/- SE]: 25.4 +/- 1.2 yr; BMI: 22.3 +/- 1.0 kg/m2). As expected, AG induced marked increase (p < 0.01) in circulating GH, PRL, ACTH and cortisol levels. AG administration was also followed by a decrease in insulin levels (-285.4 +/- 64.8 mU*min/l; p < 0.05) and an increase in plasma glucose levels (1068.4 +/- 390.4 mg*min/dl; p < 0.01). UAG alone did not induce any change in these parameters. UAG also failed to modify the GH, PRL, ACTH and cortisol responses to AG. However, when UAG was co-administered together with AG, no significant change in insulin (-0.5 +/- 40.9 mU*min/l) and glucose levels (455.9 +/- 88.3 mg*min/dl) was recorded anymore, indicating that the insulin and glucose response to AG has been abolished by UAG. In conclusion, non-acylated ghrelin does not affect the GH, PRL, and ACTH response to acylated ghrelin but is able to antagonize the effects of acylated ghrelin on insulin secretion and glucose levels. These findings indicate that unacylated ghrelin is metabolically active and is likely to counterbalance the influence of acylated ghrelin on insulin secretion and glucose metabolism. As GHS-R1a is not bound by unacylated ghrelin, these findings suggest that GHS receptor subtypes mediate the metabolic actions of both acylated and unacylated ghrelin.

Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity.

We investigated the metabolic actions of ghrelin in humans by examining the effects of acute administration of acylated ghrelin, unacylated ghrelin, and the combination in eight adult-onset GH-deficient patients. We followed glucose, insulin, and free fatty acid concentrations before and after lunch and with or without the presence of GH in the circulation. We found that acylated ghrelin, which is rapidly cleared from the circulation, induced a rapid rise in glucose and insulin levels. Unacylated ghrelin, however, prevented the acylated ghrelin-induced rise in insulin and glucose when it was coadministered with acylated ghrelin. Surprisingly, the injection of acylated ghrelin induced an acute increase in unacylated ghrelin and therefore total ghrelin levels. Finally, acylated ghrelin decreased insulin sensitivity up to the end of a period of 6 h after administration. This decrease in insulin sensitivity was prevented by coinjection of unacylated ghrelin. This combined administration of acylated and unacylated ghrelin even significantly improved insulin sensitivity, compared with placebo, for at least 6 h, which warrants studies to investigate the long-term efficacy of this combination in the treatment of disorders with disturbed insulin sensitivity.

Affinity of human growth hormone-releasing factor (1-29)NH2 analogues for GRF binding sites in rat adenopituitary.

Previous research on growth hormone-releasing factor analogues has used pituitary cell culture assay systems to evaluate in vitro their biological activity. However, binding assay systems in which receptor affinity and peptide stability can be assessed independently have been lacking so far. Since we have recently developed a sensitive GRF binding assay with [125I-Tyr10]hGRF(1-44)NH2, this method was applied to structure-affinity studies as a first step of screening GRF analogues. Acylation of the N-terminus of hGRF(1-29)NH2 generally decreased its affinity (relative affinity to hGRF(1-29)NH2 (RA), 26-85%). Replacement of the C-terminal carboxamide by a free carboxylic function decreased affinity likely by diminishing its proteolytic stability (RA, 57%). Removal of Tyr1, Ser9, Lys12, Val13, Gly15, Gln16, or Lys21 drastically decreased its affinity (RA, less than 3%). Multiple amino acid deletions in the segment 13-21 of hGRF(1-29)NH2 also led to a loss of affinity as did replacing segment 13-15, 16-18, or 19-21 by an octanoyl moiety (RA, less than 1%). Removal of Asn8, Gln24, Asp25, Ile26, Met27, and Ser28 or Arg29 had less effect on GRF receptor affinity (RA, 5-33%). Removal of Met27 or Ser28 only slightly affected hGRF(1-29)NH2 affinity (RA, 62-78%). Altogether, these results indicate that the amino acids contained in the segment 13-21 are more important than those of 24-29 to insure high affinity receptor binding or to maintain an optimal conformation to allow GRF binding.

Differential effects of GH stimulation on fasting and prandial metabolism and plasma IGFs and IGF-binding proteins in lean and obese sheep.

The effect of body condition per se on plasma IGFs and IGF-binding proteins (IGFBPs) and the whole-body metabolic responses to recombinant DNA-derived bovine GH (rbGH) in both the fed and the fasted state were determined in lean and dietary obese sheep (n = 6/group). Sheep at zero-energy balance and equilibrium body weight were injected s.c. for 12 days with 100 micrograms/kg rbGH immediately before their morning feeding. Before GH treatment, fasting plasma concentrations of insulin (17.0 +/- 1.9 vs 7.5 +/- 0.7 microU/ml), IGF-I (345 +/- 25 vs 248 +/- 10 ng/ml), glucose (52.6 +/- 1.1 vs 48.3 +/- 0.7 mg/dl), and free fatty acid (FFA) (355 +/- 45 vs 229 +/- 24 nmol/ml) were greater (P < 0.05) and those of GH (1.1 +/- 0.2 vs 2.6 +/- 0.3 ng/ml) were lower (P < 0.05) in obese than in lean sheep. Fasting concentrations of IGF-II and glucagon were not affected (P > 0.05) by obesity. GH concentrations were increased equivalently by 6-9 ng/ml in lean and obese sheep during GH treatment. GH caused an immediate and a marked fivefold increase in the fasting insulin level in obese sheep but only minimally affected insulin concentration in lean sheep. The increment in fasting glucose during GH treatment was greater (P < 0.05) in obese (8-12 mg/dl) than in lean (2-5 mg/dl) sheep. Frequent measurements in the first 8 h after feeding and injection of excipient (day 0) or the first (day 1) sixth (day 6) and twelfth (day 12) daily injection of GH showed that prandial metabolism in both groups of sheep was affected minimally by GH. However, GH treatment on day 1 (not days 6 or 12) acutely attenuated the feeding-induced suppression of plasma FFA in both groups of sheep and this effect was significantly greater in obese than in lean sheep. Although obese sheep were hyposomatotropic, the basal and GH-induced increases in plasma IGF-I concentrations were greater (P < 0.05) in obese than in lean sheep. Plasma IGF-II was unaffected by obesity and was not increased by GH stimulation. Western ligand blotting showed that IGFBP-3 accounted for approximately 50-60% of the plasma IGF-I binding capacity in sheep respectively both before and during GH treatment. Basal plasma levels of IGFBP-2 were lower (P < 0.05) and those of IGFBP-3 greater (P < 0.05) in obese compared with lean sheep. GH increased the level of IGFBP-3 equally in lean and obese sheep, but suppressed the expression of IGFBP-2 more (P < 0.05) in lean than in obese sheep. We concluded that the diabetogenic-like actions of GH in sheep were exaggerated markedly by obesity, and were expressed more during the fasted than the fed states. The effects of GH stimulation on the endocrine pancreas may be selective for beta-cells and preferentially enhanced by obesity. GH regulation of IGF-I and the IGFBPs differs in lean and obese sheep.

Cellular mechanism of caprylic acid-induced growth hormone suppression.

To determine the dynamic secretory pattern of growth hormone (GH) in the presence of free fatty acids (FFA), we studied the effect of caprylic acid on basal and rGRF(1-29)NH2 (GRF)-induced GH secretion in acutely dispersed and perifused rat pituitary cells. At a concentration of 3.0 mmol/L, caprylic acid inhibited both basal (P less than .05) and GRF-stimulated GH secretions (P less than .01), except when the maximal (near the EC100) GRF concentration of 100 pmol/L was used. Lower concentrations of caprylic acid such as 0.3 and 1.0 mmol/L significantly inhibited, in a concentration-dependent manner, GH secretion induced by a 6.25-pmol/L GRF. However, at a GRF concentration of 25 pmol/L, this inhibitory effect was abolished. The time-course of GH response to GRF was similar in both control and caprylic acid-treated cells. To elucidate the mechanism(s) of action of the caprylic acid-induced blockade of GH secretion, in 3-day cultured rat pituitary cells, the effect of caprylic acid on basal and GRF-stimulated GH and 3',5'-cyclic adenosine monophosphate (cAMP) release. We also tested its effect on the Ca2+ ionophore, A23187-induced GH release. Caprylic acid (0.3 to 3.0 mmol/L) significantly inhibited basal GH release and GRF- or A23187-induced GH secretion. Furthermore, it decreased both basal and GRF-stimulated cAMP release (P less than .05). In addition, the effect of caprylic acid on rGRF(1-29)NH2 affinity to GRF pituitary binding sites was determined using [125I-Tyr10]hGRF(1-44)NH2 as radioligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.

Effect of aging on growth hormone-induced insulin-like growth factor-I secretion from cultured rat chondrocytes.

The physiological roles of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in adult other than their effects on tissue growth is to maintain the integrity of the organism. It has been proposed that reduced availability of both hormones in late adulthood may contribute to the initiation of the major alterations and senescent changes in body composition that characterize normal human aging. Since accumulated evidence points to a direct interplay of GH with chondrocytes in cartilage, we determined in the present study the effect of aging on both basal and GH-stimulated IGF-I production from rat cultured chondrocytes. Namely, we investigated the effect of 0, 10 and 100 ng/ml of growth hormone on IGF-I levels during 1, 2, 4 and 8 days in monolayer cultured costal chondrocytes from 2-, 6-, 14- and 18-month-old rats. Measurement of IGF-I levels was done by a radioimmunoassay following a validated formic acid-heating-acetone extraction procedure. In 6- and 14-month-old rat chondrocytes, basal IGF-I secretion was higher than that of the 2-month-old control rats. In 18-month-old rat chondrocytes, basal IGF-I secretion was lower than in any other age group. Whereas in 2-, 6- and 14-month-old rat chondrocytes, GH induced a dose-related IGF-I response which was highly significant on day 8, the 18-month-old rat chondrocytes no longer responded to GH treatments. Our results suggest that the decrease in IGF-I production from cultured rat chondrocytes could be related to the blunted GH secretion in the course of aging. Therefore, GH availability in the course of aging appears to be a determinant factor in tissue responsiveness and underscores the hypothesis that GH replacement could present a therapeutic potential against the aging senescent changes.

Age-related serum levels of insulin-like growth factor-I, -II and IGF-binding protein-3 following myocardial infarction.

Aging retards the repair process by decreasing hormone secretion from the somatotrophic axis, which plays a major role in tissue reconstruction after injury. The aim of this study was to determine the effect of aging on serum insulin-like growth factor-I (IGF-I), IGF-II and IGF-binding protein-3 (IGFBP-3) levels following myocardial infarction (MI). For four consecutive days, we monitored the variation of serum IGF-I, IGF-II and IGFBP-3 concentrations in 26 patients aged 19-71 years who were diagnosed with MI. Serum IGF-I, IGF-II and IGFBP-3 were measured daily by double antibody radioimmunoassay. Daily serum IGF-I concentrations showed a significant negative correlation with age (r = -0.528, P< 0.001). Total serum IGF-I was significantly (P = 0.002) higher in the younger age group (patients under 50 years) compared to the older group (50 years and over); 206 +/- 16 ng/ml vs 136 +/- 12 ng/ml. During this investigation, younger patients (under 50 years) showed no significant daily variations in IGF-I levels compared to older patients (50 years and over) who presented a significant decline (P = 0.012). Total serum IGF-II in both groups decreased significantly with time. Total serum IGFBP-3 in the younger age group was significantly higher (P = 0.046) than in the older age group (3.42 +/- 0.18 microgram/ml vs 2.95 +/- 0.13 microgram/ml). MI patients in both groups showed significantly lower IGF-I and IGF-II (IGFs) with higher IGFBP-3 compared to age- and sex-adjusted levels of normal adults (controls). The present results confirm that age and cardiac condition affect IGFs and IGFBP-3 levels. We are inclined to believe that older patients with a cardiac condition are less able to maintain their blood IGF-I levels during the recovery period compared to younger patients. Given the biological impact of IGF-I on regeneration, this could explain why older patients take longer to recover and heal poorly in comparison to younger patients.

The use of pigs as an animal model to evaluate the efficacy, potency and specificity of two growth hormone releasing factor analogues.

In 1982, Guillemin et al reported the isolation of the human (h) growth hormone (GH) releasing factor (GRF) from a pancreatic tumour in an acromegalic patient. Since then, work to develop potent GRF analogues has been widespread and the rat has been the main animal model used. The aim of the present study was to compare the efficacy, potency and specificity of two GRF analogues with those of the native GRF(1-29)NH(2)using pig (p) as the animal model. Two analogues, Al ([His(1), D-Ala(2), Ala(8,9,15,17), D-Arg(29)] hGRF(1-29)NH(2)) and A2 ([D-Ala(2), Ala(8,9,15,17), D-Arg(29)] hGRF(1-29)NH(2)) were compared with the h or pGRF(1-29)NH(2). Five studies were designed using 28-48 kg BW growing barrows. Results showed that the two GRF analogues were more potent than the native GRF molecule, were highly specific, were active for long periods of time and were able to induce changes in body composition similar to those reported with GH or other GRF analogues. Because of the similarity between swine and human species with respect to the amino acid sequence of GRF and to the physiology, secretion and effects of GH, it can be proposed that the pig could be used as a pre-clinical animal model to study and test new GRF molecules over short and long periods of time.

Characterization of [125I-Tyr10]human growth hormone-releasing factor (1-44) amide binding to rat pituitary: evidence for high and low affinity classes of sites.

A sensitive binding assay was developed to determine binding characteristics of commercially available [125I-Tyr10]human growth hormone-releasing factor (hGRF) (1-44)NH2 in rat pituitary using 0.1 gland homogenate (70-75 micrograms protein) per incubation tube. Under standard assay conditions, addition of 5 mM EDTA efficiently prevented the degradation of both human and rat GRF for at least 3 h. Association of the ligand was time-dependent: equilibrium was reached within 30 min of incubation at 23 degrees C and remained stable for an additional 150 min (K1 = 5.01 +/- 0.86 nM-1.min-1). Specific binding increased linearly with the amount of protein present in the assay, from 15 to 170 micrograms per incubation tube. This binding was reversible, dissociation occurring almost completely after a 120-min period (K-1 = 8.13 +/- 0.29 x 10(-3) min-1). A concentration of 5-10 mM Mg2+ was required for optimal specific binding whereas 50 mM Mg2+ or monovalent cations such as Na+, K+, Li+ decreased it. Scatchard analysis of cold saturation studies by the Ligand program statistically revealed the presence of two distinct classes of binding sites; the first was of high affinity (0.68 +/- 0.11 nM) and low capacity (140 +/- 22 fmol/pituitary), the second was of lower affinity (590 +/- 347 nM) and higher capacity (38.7 +/- 18.7 pmol/pituitary). Similar values were obtained with various bovine serum albumin (BSA) concentrations and when using crude or washed pituitary homogenates, suggesting that the second low affinity site was not BSA or a soluble protein from the homogenate.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I concentration in Holstein female cattle: variations with age, stage of lactation and growth hormone-releasing factor administration.

Plasma insulin-like growth factor-I (IGF-I) concentrations were monitored in Holstein females through different periods of their growth, lactation and after acute or chronic growth hormone-releasing factor (GRF) administration. Plasma samples were radioimmunoassayed using a human IGF-I antibody after a 24 hr incubation in a HCl(.1N)-glycine(.2M) buffer (pH 2). In a first study, IGF-I concentrations were measured in Holstein females of different ages and(or) stages of lactation (n = 6 per group). The IGF-I concentrations in newborn calves (102.0 +/- 11.3 ng/ml) markedly decreased (P less than .01) in 1 mo old animals (50.2 +/- 7.1 ng/ml), then increased (P less than .01) to 137.0 +/- 5.1 and 137.4 +/- 11.0 ng/ml in 6 and 10 mo old heifers, respectively. In dairy cows, IGF-I concentrations were low 24 hr post-partum (44.7 +/- 7.6 ng/ml) and then increased (P less than .05) to remain stable throughout lactation (91.3 +/- 4.9, 92.8 +/- 12.9, 96.1 +/- 7.6, 90.7 +/- 8.8 ng/ml at 2, 3, 6 and 9 mo of lactation, respectively). There was a further increase (P less than .05) to 113.7 +/- 3.1 ng/ml during the dry period. In a second trial, blood samples were collected from lactating dairy cows every 2 hr for 24 hr following a sc injection of saline (n = 4) or human (h) GRF (1-29)NH2 (10 micrograms/kg BW, n = 4). The IGF-I peak concentration was reached on average 10 hr after the GRF injection and was higher (P less than .01) in treated cows than in control cows (135.4 vs 86.9 +/- 16.2 ng/ml). In the last trial, daily sc injections of 10 micrograms of hGRF(1-29)NH2 per kg BW to dairy cows (252 days of lactation) for 57 days, which increased milk production by 14% (2 kg/day), also increased (P less than .01) IGF-I concentration: 127.1 +/- 5.3 and 118.0 +/- 1.6 vs 90.7 +/- 4.7 and 96.0 +/- 5.0 ng/ml on days 29 and 57 of treatment for treated (n = 9) and control (n = 8) cows, respectively. Thus, the IGF-I concentration in dairy cattle varies with age and stage of lactation, and is increased by GRF administration in lactating dairy cows.

Ontogeny of insulin-like growth factors (IGF), IGF binding proteins, IGF receptors, and growth hormone receptor mRNA levels in porcine pancreas.

We examined the ontogeny of mRNA levels of IGF-I and -II, IGF type 1 (IGFI-R) and type II receptors (IGFII-R), IGF binding protein-1 and -3 (IGFBP-1 and -3), GH receptor (GHR), and tissue concentrations of IGF and IGFBP in the pancreas of pigs. Tissues were collected from fetuses at 90 and 110 d of gestation and from pigs at 1, 21, 90 and 180 d of age. Northern blots were performed using total RNA hybridized with 32P-labeled cDNA probes (human IGF-I and human IGFI-R) and cRNA probes (rat IGF-II, human IGFII-R, human IGFBP-1, pig IGFBP-3, and pig GHR). There were two accelerated growth stages of the pancreas: the first one at 90 d of fetal life, which is characterized by cell hyperplasia (high ratio of DNA to body weight), and the second one at postnatal 90 d, which is attributed to cell hypertrophy (high ratios of pancreatic weight, RNA, and protein to DNA). The level of IGF-II mRNA and its tissue concentration were predominant during fetal life and low thereafter. The IGF-I mRNA level was high during fetal and early postnatal life and decreased thereafter. Messenger RNA levels of IGFI-R, IGFBP-3, and GHR and concentrations of IGFBP-1 and -2 were abundant during fetal and early postnatal life. In conclusion, IGF may be involved in various physiological periods of pancreatic development in pigs.

Long-term growth hormone-releasing factor administration on growth hormone, insulin-like growth factor-I concentrations, and bone healing in the Beagle.

Twelve 11 month old male Beagles were assigned to two treatment groups: a control group (saline) and a group receiving human growth hormone (GH)-releasing factor (hGRF) [1-29]NH2 (25 micrograms/kg, SC, TID). Treatment was started 6 days prior to surgery (day 1) and continued until necropsy (3 dogs per group/day) on d 29 or 58. Two porous polyethylene rods were surgically implanted on the lateral diaphysis of the femoral shaft and a 3 mm hole was drilled through the cortex between the two implants of each dog on day 1. Blood and urine were collected on d -6, 27 and 56. Human GRF injections produced a significant (P < 0.05) increase in GH release following each injection. An increase in GH response was also observed (P < 0.05) over time. The concentration of insulin-like growth factor-1 (IGF-1) increased for 5 weeks and then reached a plateau. None of the hematologic or urine measured parameters was affected by the treatment (P > 0.05). Albumin, calcium, and protein concentrations were higher (P < 0.05) on d 27 and 56 in GRF-treated animals. Histological sections of the onlay sites showed that bony ingrowth tended to be greater into the porous polyethylene material in GRF-treated animals than the controls at d 28 and 57, while no difference was observed in the degree of periosteal bone formation around the implants at either time period (P > 0.05). Bone formation into the cortical defect was greater in the GRF-treated dogs when compared to controls at day 57 only. In conclusion, chronic hGRF [1-29]NH2 treatment in Beagle dogs produced an increased GH response over time and increased IGF-1 concentrations. It also appeared to promote bony ingrowth into a porous polyethylene onlay and into a bony deficit.

Nitrogen balance and mineral excretion in growing male pigs injected with a human growth hormone-releasing factor analog.

A human growth hormone-releasing factor analog ([Desamino-Tyr1,D-Ala2,Ala15] hGRF(1-29) NH2) has been reported to reduce feed intake and increase growth and feed efficiency in a dose-dependent manner in growing pigs. The aim of this study was to determine the effect of this analog on nitrogen (N) balance and mineral excretion. Fifteen castrated male Yorkshire x Landrace pigs (45.9 +/- 1.4 kg) were randomly allotted to 2 groups: control (saline, n = 7) and GRF (6.66 micrograms/kg sc, TID, n = 8). The animals were injected for 20 consecutive days: feces and urine were collected during the last 10 d of injection. The animals had free access to water and food until satiety (approximately 15 min) at 07:00, 11:00, 15:00, 19:00, 23:00 and 07:00 h. The diet consisted of a hog fattening ration (18.0% crude protein). Blood samples were collected on the last day of the study by venipuncture. This analog increased (P < 0.05) insulin-like growth factor-1 and glucose serum concentrations and decreased (P < 0.05) serum urea nitrogen concentration and feed intake. The GRF-treated animals ingested less N, excreted less N in urine and feces to retain a similar amount of N than controls. The apparent coefficient of digestibility of the N has been slightly increased (P < 0.05) by GRF. Urinary excretion of P, K, and Cl decreased (P < 0.01) with GRF treatment. In conclusion, this GRF analog increased N digestibility and retention relative to N ingestion and reduced urinary N, P, K, and Cl excretion.

Ontogeny of IGFs and IGFBPs mRNA levels and tissue concentrations in liver, kidney and skeletal muscle of pig.

As far as we know, there is no available information about ontogenic changes of tissue concentrations of IGF-I and II and IGFBPs in large mammals. Serum, liver and kidney levels of IGFs and IGFBPs were examined in fetuses at 90 and 110 days of gestation and in pigs at 1d, 3 wk, 3 mo and 6 mo of age. Ontogeny of mRNA levels of IGFs, IGF type I and type II receptors (IGFI-R and IGFII-R), IGFBP-1 and -3 (IGFBPs) and growth hormone receptor (GHR) were also examined by Northern blot analysis in liver, kidney and skeletal muscle of pig. Serum IGF-I, IGF-II and IGFBP-3 concentrations were low during the fetal life and increased after birth. The highest level of IGF-II mRNA was found in fetuses for all studied tissues. In the liver, IGF-I mRNA level and its protein content peaked at 3 wk of age. The highest IGF-II concentration was found at 1d and 3 wk of age. The IGFII-R mRNA remained at a constant level during the whole development period. The most abundant IGFBP-1 mRNA and its protein content were found at birth. The level of IGFBP-2 was high during fetal and early postnatal life. The IGFBP-3 content was relatively low in fetuses and reached the highest level after 3 wk of age. In the kidney, IGFs, IGFBP-3, IGFI-R and IGFII-R as well as GHR mRNA levels were relatively high during the fetal and early postnatal life. The IGFs concentrations were the highest in newborns. In the skeletal muscle, IGFs, IGFBP-3 and IGFI-R mRNA levels decreased with advancing age. During the postnatal life, the high IGFs concentrations in the liver and the kidney correspond to fast growth periods of these organs.

Growth hormone response induced by synthetic human growth hormone-releasing factor (1-44) in healthy dogs.

The growth hormone (GH) secretory response to varying doses (1, 5 and 25 micrograms/kg) of intravenous (i.v.) human GH releasing factor (GRF) (1-44) was measured in four, 16-month-old female dogs and compared to the GH response to placebo (vehicle solubilizing GRF) and standard clonidine test (10 micrograms/kg i.v.). The three doses of GRF had no apparent clinical side effects and were capable of eliciting a rapid increase in the mean plasma GH level. The rise in plasma GH occurred within 5 minutes with a peak response at 15-30 minutes. However inter-subject variability of the GH response was large in terms of magnitude and time course. By analysis of the areas under the response curves it was shown that GRF 1-44 was as effective in stimulating GH release as clonidine and that a dose of 1 microgram/kg GRF 1-44 could be a good choice for use as a standard test dose in the dog.