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.

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.

Healing of experimental aneurysms. II: Platelet extracts can increase the thickness of the neointima at the neck of treated aneurysms.

PURPOSE: To assess the effect of platelet extracts (PE) on neointima formation following gelfoam packing of experimental porcine aneurysms. A strategy involving the local delivery of platelet growth factors may potentially improve long term results of endovascular treatment of aneurysms. METHODS: Bilateral lateral wall common carotid aneurysms were constructed on 30 pigs. A collagen sponge containing a PE rich in growth factors was used to pack one aneurysm with the controlateral lesion being embolized with a sponge containing NaCl 0.9% (22 animals). In 8 animals, a control sponge was used on both sides. Animals were sacrificed at 1, 2, 3, 4 and 9 weeks and the thickness of the neointima covering the neck of PE-treated aneurysms was measured in 5 locations for each lesion at 2 and 3 weeks and compared with the control aneurysm of the same animal. Morphometric data was analysed using the paired Student's t-test. RESULTS: The thickness of the neointima was significantly increased in lesions treated with PE as compared to control lesions at 2 weeks (p = 0.008, n = 9). There was no significant difference at 3 weeks (p = 0.99, n = 9). There was no significant difference between lesions of control animals (p = 0.95, n = 8). CONCLUSION: PE rich in growth factors can increase the thickness of the neointima at the neck of treated experimental porcine aneurysms at 2 weeks, but had no effect at 3 weeks. This accelerated neointimal formation may have some value in improving healing following endovascular treatment. This hypothesis could not be supported with this experimental model which has a spontaneous tendency to heal. Further studies using an animal model which reproduces the clinical problem of recurrences may help to define the role of the local delivery of growth factors in combination with coils in a strategy designed to improve results of endovascular treatment.

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.

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.

IGF and IGF-binding protein system in the synovial fluid of osteoarthritic and rheumatoid arthritic patients.

Various arthritic disorders result from a disruption of the equilibrium between the synthesis and degradation of tissue matrix macromolecules. Growth factors, particularly insulin-like growth factor-I (IGF-I), are believed to play an important role in maintaining this equilibrium. In this study, we determined the levels of IGF-I, IGF-II, and characterized and measured the amount of IGF-binding proteins (IGFBPs) in the synovial fluid (SF) of osteoarthritis (OA), rheumatoid arthritis (RA) patients and normal individuals. Furthermore, we characterized the IGFBP found in these SFs. The levels of IGF-I, IGF-II and IGFBP-3 were determined by specific radioimmunoassays (RIAs). IGFBP identification and measurement were carried out using the Western ligand blot (WLB) technique, and characterization performed by Western immunoblot. IGFBP-3 proteolysis was analyzed by autoradiography after incubation of SF with radiolabeled IGFBP-3. Results showed a statistically significant increase (P < 0.001) in the IGF-I level in arthritic SF vs normal controls; 75 +/- 11 ng/ml and 82 +/- 11 ng/ml were recorded for RA (N = 8) and OA (N = 10), respectively, whilst normal controls (N = 9) were at 19 +/- 7 ng/ml. No difference in the level of IGF-II was recorded between the three groups studied. Human SF demonstrated the presence of IGFBP-1, -2, -3 and -4, but not that of IGFBP-5 and -6. The level of IGFBP-3 tested either by WLB or RIA was significantly higher (P < 0.001) in RA and OA patients. Moreover, a statistical and positive correlation between the levels of IGF-I and IGFBP-3 was noted. WLB analysis indicated that the amount of IGFBP-1 did not vary among the groups. The levels of IGFBP-2 and -4 were significantly increased (P < 0.02) solely in the RA SF. Further experiments demonstrated that a limited IGFBP-3 proteolysis occurred in human SF. Moreover, the ratio of total IGF over total bioactive IGFBPs was lower in RA (P < 0.05), and to a lesser extent in OA than normal specimens. This study showed the presence of four IGFBPs (1 4) in human SF for which the IGFBP-2, -3 and -4 were enhanced in arthritic fluid. Importantly, although proteolysis occurred in the SF, an increased amount of bioactive IGFBPs were present in arthritic SF, which may affect the bioavailability of IGF-I within the articular tissues.

Local induction of an insulin-like growth factor binding protein-3 degrading protease activity in the course of wound healing.

Proteases that reduce insulin-like growth factor binding protein-3 affinity for insulin-like growth factor-I have been found in various biological fluids from human beings and rats. The aim of this study was to assess the local and systemic role of insulin-like growth factor binding protein-3 proteases in the course of wound healing. Six rats had polyvinyl alcohol sponges implanted subcutaneously. Wound fluid and serum were collected 3 days after wounding. Gel filtration experiments showed that insulin-like growth factor-I was present as a 150 kDa complex in both serum and wound fluid. However, insulin-like growth factor binding protein-3 measured by Western ligand blotting was virtually absent in wound fluid. Co-incubation of serum and wound fluid resulted in an ethylenediamine tetraacetic acid-inhibitable degradation of serum insulin-like growth factor binding protein-3, suggesting the presence of an insulin-like growth factor binding protein-3 degrading activity in wound fluid. Incubation of ((125)I)-labeled insulin-like growth factor binding protein-3 in wound fluid and serum showed a rapid and time-dependent proteolysis of insulin-like growth factor binding protein-3 in wound fluid with metabolites similar to those generated by human term pregnant serum. No sign of insulin-like growth factor binding protein-3 degrading activity was observed in rat-serum. In conclusion, there is an insulin-like growth factor binding protein-3 proteolytic activity in wound fluid, and it is hypothesized that this activity results in a localized increase in insulin-like growth factor-I bioactivity.

Inhibition by rapamycin of PDGF- and bFGF-induced human tenon fibroblast proliferation in vitro.

PURPOSE: Fibrosis of subconjunctival tissues is a major cause of bleb failure following glaucoma filtration surgery. The aim of the present investigation was to demonstrate the effect of Rapamycin, a clinically relevant macrolide antibiotic with potent immunosuppressive properties, on human Tenon fibroblast proliferation induced by platelet-derived growth factor and basic fibroblast growth factor. METHODS: Primary Tenon fibroblast cultures were derived from patients undergoing trabeculectomies or routine cataract extractions. Rapamycin was added in concentrations of 0.1-100 ng/ml with or without 3-30 ng/ml of porcine platelet-derived growth factor or of human recombinant basic fibroblast growth factor. Two days after treatment, the cells were examined and counted. The results were expressed as the percent of cell growth in treated culture relative to its untreated control. RESULTS: Rapamycin was not cytotoxic at any of the concentrations tested. Inhibition of platelet-derived growth factor-induced Tenon fibroblast proliferation occurred with all doses of Rapamycin, the most marked effect being observed with 30 ng/ml (60% inhibition, p < 0.001). In contrast, optimal inhibition of basic fibroblast growth factor-induced proliferation was only 37% (p < 0.01), achieved with 10 ng/ml of the peptide. CONCLUSION: Rapamycin potently inhibits platelet-derived growth factor-induced fibroblast proliferation in vitro without any apparent cytotoxicity. It may eventually prove to be a useful adjunct to glaucoma filtration surgery.

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.

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.

Age-related changes in peptide-23/pancreatitis-associated protein and pancreatic stone protein/reg gene expression in the rat and regulation by growth hormone-releasing hormone.

Peptide-23 is a 16-kilodalton protein secreted by rat pituitary cells that was first identified because it was regulated by GRF and somatostatin in a similar fashion to GH. Cloning of peptide-23 complementary DNA revealed that it is identical to pancreatitis-associated protein (PAP) and a member of the c-lectin gene family. We examined the expression of peptide-23/PAP and a structurally related protein, pancreatic stone protein (PSP/reg), in the rat gastrointestinal tract. Here we report age-related changes in the expression and GRF regulation of peptide-23. Both peptide-23/PAP messenger RNA (mRNA) and PSP/reg mRNA were virtually undetectable in the small intestine of newborn and 1- and 2-week-old rats. A dramatic increase in the expression of both genes was seen at the time of weaning in the third week postpartum. The abundance of both of these mRNA decreases after 3 and 6 months of age. Peptide-23/PAP mRNA is most abundant in the ileum, whereas PSP/reg is maximally expressed in the pancreas and duodenum. Human GRF analog pellets were implanted sc into adult male rats for 2 weeks to study the chronic effects of GRF on the expression of these genes. Both peptide-23/PAP and PSP/reg mRNA levels in duodenum and jejunum were increased in these rats compared with levels in control rats. However, no increase in peptide-23/PAP mRNA in response to GRF treatment was seen in the ileum, where the level of expression of this gene is very high, and GRF had no effect on peptide-23/PSP expression in the heart, pituitary, or hypothalamus, where expression is normally undetectable. In situ hybridization was used to localize peptide-23/PSP in the small intestine and pancreas of GRF-treated rats. An increase in peptide-23/PAP mRNA was restricted to acinar cells close to islets, whereas little expression was seen in acinar cells distant from islets, suggesting that either peptide-23/PAP may have some paracrine action on the islets, or alternatively, an islet-derived factor may function as a paracrine modulator of peptide-23/PAP expression. These data demonstrate that GRF modulates peptide-23/PAP expression in the gastrointestinal tract in a similar fashion to that previously reported for pituitary cells in primary culture.

The effect of feed intake and growth hormone-releasing factor on lactating dairy cows.

Twenty-four lactating dairy cows, averaging 30.0 kg/d of milk and 159 d of lactation, were used to study the effect of feed intake and growth hormone-releasing factor in a 2 x 2 factorial arrangement. For the first 10-d period, cows had free access to a TMR and received a fixed amount of high moisture corn, protein supplement, and hay. In the second 10-d period, 12 cows were maintained on this high intake, and 12 cows received 70% of their previous intake (low intake). During the following 10-d period, each intake group was divided, and each of two subgroups (n = 6) received twice daily s.c. injections of saline or growth hormone-releasing factor (10 micrograms/kg of BW per injection). Feed restriction decreased milk production by 24%. Milk production increase was not different following growth hormone-releasing factor treatment for cows maintained at high intake (4.6 kg/d) or low intake (3.4 kg/d). Feed restriction increased concentration of growth hormone but did not affect IGF-I concentration. Growth hormone-releasing factor increased IGF-I concentration similarly for both intake groups but increased concentrations of insulin and IGF-binding proteins-1 and -3 only in the high intake group. Low intake did not affect growth hormone, IGF-I, or milk responses to growth hormone-releasing factor, but suppressed the increase in concentrations of insulin and IGF-binding proteins-1 and -3 following treatment with growth hormone-releasing factor for cows on high intake.

Increased insulin-like growth factor 1 production by human osteoarthritic chondrocytes is not dependent on growth hormone action.

OBJECTIVE: To investigate insulin-like growth factor 1 (IGF-1) production in normal and osteoarthritis (OA) chondrocytes and to further examine the role of growth hormone (GH) in adult human cartilage and, in particular, in diseased tissue. METHODS: IGF-1 production was measured with a radioimmunoassay. Binding assay, Northern blot, and reverse transcriptase polymerase chain reaction (RT-PCR) techniques were used for GH receptor (GHR) detection. The biological response to GH was estimated via IGF-1 production. RESULTS: We observed that basal levels of IGF-1 production were significantly higher in OA chondrocytes than in normal cells (P < 0.005). Adult human chondrocytes, however, were unresponsive to GH stimulation with regard to IGF-1 production, as shown in dose-response (0-1,000 ng/ml) and time-course (days 1-8) studies. In addition, no specific 125I-GH binding was detected in either cell type. Northern blot analysis revealed a 5.5-kb GHR messenger RNA (mRNA) species, but semiquantitative RT-PCR revealed no difference in GHR mRNA expression by normal and OA chondrocytes. CONCLUSION: This study indicates that the elevated synthesis of IGF-1 by adult human OA chondrocytes occurs through a GH/GHR-independent mechanism, suggesting that other factors are capable of controlling local IGF-1 production in these cells.

Long-term effects of human growth hormone-releasing hormone and photoperiod on hormone release and puberty in dairy heifers.

Forty-eight Holstein dairy heifers (98.9 kg BW; 3 mo old) were subjected for 246 d to twice-daily s.c. injections of saline (CTL) or human growth hormone-releasing hormone (GRH; 5 micrograms/kg BW) and to photoperiods of 8 h of light (L): 16 h of dark (D) or 16L:8D according to a 2 x 2 factorial arrangement of treatments. Jugular blood samples were collected from 16 heifers at 3, 4, 8, and 11 mo of age to monitor prolactin, growth hormone, and estradiol-17 beta. Plasma progesterone concentrations were monitored weekly in all heifers as an index of puberty (> 1 ng/mL). Growth hormone release was induced by GRH (P < .001) throughout the trial; area under the GH curve (AUC) averaged 1,582 vs 3,643 ng.min-1.mL-1 in CTL vs GRH heifers. However, GRH-induced GH response was less (P < .05) after the second daily injection. There was also an interaction (P = .08) between GRH, photoperiod, and days of treatment on GRH-induced GH response; AUC was greater in GRH-16L:8D than in GRH-8L:16D heifers at 3 mo but less at 8 mo of age. The PRL concentrations were similar for both photoperiods at 3 mo (36.4 vs 41.7 ng/mL) and 8 mo (16.2 vs 12.8 ng/mL) of age but were greater in 16L:8D vs 8L:16D heifers at 4 mo (18.4 vs 39.3 ng/mL) and 11 mo (26.3 vs 44.1 ng/mL) of age (photoperiod x day interaction, P < .001). Photoperiod of 16L:8D vs 8L:16D reduced (P < .01) weight at puberty in CTL heifers (251 vs 303 kg BW) and to a lesser extent in GRH-treated heifers (271 vs 284 kg BW; GRH x photoperiod interaction, P = .10). In conclusion, GH response is maintained throughout 8 mo of GRH treatment, and a 16L:8D photoperiod will reduce age and weight at puberty in heifers. Furthermore, refractoriness to photoperiod-induced PRL changes was detected.

Effects of GRF with or without a SRIF antiserum on GH, IGF-1, thyroxin, cholecystokinin, gastrin and metabolite concentrations in growing rats.

This study was undertaken to examine the effects of increasing doses of rat somatocrinin (GRF) with or without a somatostatin antiserum (SS-ab) on serum hormone and metabolic concentrations, as well as serum and duodenal cholecystokinin (CCK) and antral gastrin concentrations. 24-day-old male Sprague-Dawley rats were injected twice daily s.c. (10:00 and 16:30) for 14 consecutive days with either saline or rat GRF (1-43) NH2 (4 and 20 micrograms/kg) in gelatin. Three other groups of animals received the same treatment in association with the SS-ab given i.p. every other day making up the 6 groups of 12 animals in a 2 x 3 factorial design experiment. GRF treatment increased circulating growth hormone (GH) concentrations in a dose-dependent manner, alone or in combination with the SS-ab; the SS-ab treatment alone or combined with GRF also increased GH concentrations. Total hypophyseal GH content was increased (P < 0.05) by the GRF treatment alone. Serum levels of IGF-1, acetoacetate, alpha 2 globulin and antral gastrin were all increased by the GRF treatment with plateaus observed for antral gastrin and serum IGF-1 levels at the intermediary dose of GRF. Serum concentrations of T4 were reduced at the 4 micrograms/kg dose of GRF. Serum concentrations of CCK were increased by the SS-ab treatment alone, an effect reversed by increasing doses of GRF. Rat GRF produced a dose-dependent increase and decrease of alpha 2 globulin and albumin, respectively. These data indicate that GRF, probably via its effect on GH release, influences gastrointestinal hormone levels which are implicated in gastrointestinal organ growth and digestive processes.

Human osteoarthritic chondrocytes possess an increased number of insulin-like growth factor 1 binding sites but are unresponsive to its stimulation. Possible role of IGF-1-binding proteins.

OBJECTIVE: To characterize the insulin-like growth factor 1 (IGF-1) receptor in human osteoarthritic (OA) and normal adult chondrocytes. The biologic response of chondrocytes to IGF-1 stimulation was examined, as was the presence and synthesis of IGF binding proteins (IGFBP) in these cells. METHODS: Binding studies, Northern blot, immunohistochemical analysis, and affinity cross-linking experiments were performed for characterization of the IGF receptor, and the latter method was also used for IGFBP determination. The biologic response was estimated via the incorporation of radiolabeled proline into a newly synthesized protein. RESULTS: Binding experiments revealed a single class of binding sites. The mean +/- SEM affinity (Kd) of normal chondrocytes was 1.4 +/- 0.4 nM, with 26.8 +/- 5.5 x 10(3) binding sites/cell. OA chondrocytes had a lower affinity (Kd 15.4 +/- 4.7 nM) and a higher density (1,178.3 +/- 299.5 x 10(3) binding sites/cell) compared with normal cells (P < 0.004 and P < 0.001, respectively). Immunohistochemical studies with a monoclonal antibody (MAb) against the type 1 IGF receptor (alpha IR3) showed increased staining in OA cartilage compared with normal tissue. Biologic responses of chondrocytes after IGF-1 stimulation revealed that OA chondrocytes were unresponsive, whereas a 2.5-fold increase in new protein synthesis was observed in normal cells. Competition studies in normal chondrocytes revealed that both IGF-1 and IGF-2 displaced radiolabeled IGF-1 in a comparable manner; however, insulin at high concentration weakly competes. Moreover, MAb alpha IR3 effectively blocked specific binding in normal chondrocytes (77%), but not in OA chondrocytes (26%). Northern blot and covalent cross-linking analyses revealed the specific band characteristic of type 1 receptor. With the latter technique, other bands corresponding to the IGFBPs were also detected. Comparison between normal and OA chondrocytes showed increased intensity of the IGFBP bands, particularly those corresponding to the IGFBP-3 doublet. CONCLUSION: It is shown that type 1 IGF receptor is expressed in human articular cartilage and that the level of binding sites is significantly increased in OA chondrocytes. Interestingly, despite the higher level of binding sites in OA cells, no response to IGF-1 stimulation was found in these cells. Our data suggest that this increase in specific binding may involve not only the type 1 IGF receptor but also IGFBP on the cell surface. The latter, by binding the IGF-1, will diminish the bioavailability of IGF-1 and thus prevent its anabolic action.

Insulin-like growth factor-I blood levels in severely burned patients: effects of time post injury, age of patient and severity of burn.

OBJECTIVES: Insulin-like growth factor-I (IGF-I) is a polypeptide growth factor that stimulates protein synthesis. The aims of this study were to determine (1) the effect of a severe burn on blood IGF-I levels and (2) the variables controlling IGF-I level variations during recovery of these hypermetabolic patients. PATIENTS: Eleven patients, nine men and two women (age range 22-55 years) were studied for 25 days following a severe burn (18-75% of total body surface area, mean 36%). Nitrogen balances were recorded daily and total IGF-I levels were measured every 3 days. MEASUREMENTS: IGF-I was extracted from serum using a validated formic acid-acetone methodology, then measured by a double antibody radioimmunoassay. IGF-I levels were compared to those of a reference healthy population. RESULTS: Within 24 hours following injury, IGF-I levels were low in all patients when compared to normal values for the same age range (mean +/- SEM of all patients, 131 +/- 26 micrograms/l). They remained low for the first week (days 4 and 7, 109 +/- 16 micrograms/l), then increased to reach normal values at the end of the study period (days 10-16, 144 +/- 19 micrograms/l, P = 0.005 when compared to days 4-7; days 19-25, 206 +/- 30 micrograms/l, P = 0.008 when compared to days 10-16). IGF-I levels were negatively correlated with age in the second phase of recovery only (days 10-16, r = -0.70, P < 0.05; days 19-25, r = -0.75, P < 0.01) and with severity of burn between days 19 and 25 (r = -0.62, P < 0.05). The presence of bronchial burn injury tended to lower IGF-I blood concentration (P = 0.08). Whereas IGF-I concentrations increased in the later phase of recovery, nitrogen balances did not. As a result, there was no significant correlation between these parameters. CONCLUSIONS: IGF-I levels followed a biphasic pattern in severely burned patients. They dropped dramatically in response to the traumatic shock, then increased during recovery. The degree of increase was dependent on the age of the patient and on the severity of the burn, but was not associated with an improvement in the nitrogen balance.

Age-related changes in secretion rate and post-secretory metabolism of growth hormone in swine.

The effect of age on growth hormone (GH) metabolism and GH-releasing factor (GRF)-induced GH concentrations were studied in 7 young (3 mo, 39 kg) and 7 old (30 mo, 156 kg) Yorkshire x Landrace female pigs. Jugular catheters were surgically inserted and 60 hr later total serum volume was determined. The following day, all animals were infused for 3 hr with GH (30.3 ng.min/kg B.W.) in order to calculate GH metabolic clearance rate (MCR), secretion rate (SR) and half-life (t 1/2). Two days later, 15 micrograms/kg of GRF was injected i.v. into all pigs. On a per animal basis, aging increased (P < .01) MCR (299 vs 132 ml/min), SR (714 vs 422 ng/min) and serum volume (6.6 vs 2.01), whereas t1/2 was unaltered (P > .1). Basal GH concentrations were lower in older pigs (P < .10) but the GRF-induced GH concentrations (measured as GH peak or area under the curve, AUC) were not affected by age (P > .1). Yet, when induced total GH secretion (AUC x MCR) and average total serum GH (mean GH post-injection x serum volume) were calculated per pig, these variables significantly increased between 3 and 30 mo of age. Basal IGF-I concentrations were lower in older pigs (P < .01), yet, there was a tendency (P = .10) for these pigs to show a greater IGF-I response to GH infusion. The present data therefore indicate that age alters both SR and post-secretory metabolism of GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of a culture system for porcine pituitary cells: effects of growth hormone-releasing factor and(or) somatostatin on growth hormone secretion.

The goal of the study was to establish the age-related responses of cultured porcine pituitary cells to growth hormone-releasing factor (GRF) and(or) somatostatin (SRIF). A culture system for dispersed porcine pituitary cells was validated. Pituitaries from female pigs of various ages (90 or 110 d of gestation, newborn, 3, 6, or 24 mo old) were enzymatically dispersed with collagenase and neuraminidase, plated (200,000 cells/well), and cultured for 3 d. Plated cells were then subjected to a 4-h challenge with increasing concentrations of GRF (10(-11) to 10(-8) M), SRIF (10(-9) to 10(-6) M), or 10(-8) M of each peptide with increasing concentrations of the other. Culture media were collected and assayed for growth hormone (GH). Pituitaries were pooled so that there were four replicates per age, and treatments were assigned to quadruplicate wells. Concentrations of GH in control wells (basal GH) were maximal at 110 d of gestation and decreased thereafter (P < .01) with increasing age of swine. All peptide combinations affected the GH response (P < .05) at all ages studied, yet GRF was more potent than SRIF in eliciting a response. Age had an effect (P < .05) on the GH response to any of the treatments; younger pigs (90, 110 d of gestation and newborns) had a greater response (P < .05) than older pigs (3, 6, and 24 mo), whereas 6- and 24-mo-old pigs responded similarly in all cases (P > .1).(ABSTRACT TRUNCATED AT 250 WORDS)

Digestive enzyme development in newborn piglets born of sows immunized against somatostatin and/or receiving growth hormone-releasing factor during gestation.

Thirty-eight second parity sows were either immunized (IMM) against somatostatin (SRIF) and/or injected with growth hormone-releasing factor (GRF) during gestation. Treatment effects on pancreatic, gastric and duodenal development as well as on digestive enzyme activity of piglets at birth (before suckling) or at 24 h postpartum were investigated. Birth weights of piglets were similar across treatments (p > 0.1). Weight, DNA, RNA, total protein content and enzyme activity for all three organs increased between birth and 24 h postpartum (p < 0.01), except for pancreatic RNA and chymotrypsin which decreased (p < 0.01), and protein content of the pancreas which was unaltered (p > 0.1). Gastric RNA, pancreatic weight:DNA, RNA:DNA and amylase:DNA ratios were increased in 1-day-old piglets from SRIF-IMM sows (p < or = 0.05). GRF only had significant effects (p < 0.05) on the maltase:DNA ratio, which it decreased. Yet, there were tendencies (p < 0.1) for duodenal weight, DNA and total protein content to be increased in piglets from GRF-injected dams. It is therefore apparent that major developmental changes of the pancreas, stomach and duodenum of piglets take place during the first 24 h postpartum. Injections of GRF and/or immunization against SRIF during gestation in swine also have several effects on digestive enzyme activity of neonatal pigs. Yet, the physiological implications of these early changes are not clear at the present time.