Paul Brazeau

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.

Somatostatin: A peptide with unexpected physiologic activities

Somatostatin was the first in a series of peptides found to have physiologic effects both at the periphery and in the central nervous system. A prototype of peptides with varied distribution and multiple effects, somatostatin has given us many insights into the numerous physiologic implications of varied anatomic distribution. Somatostatin was the first hormone to broaden our classic concepts of endocrinology. Although its endocrine mode of action at the hypothalamo-pituitary axis is well demonstrated, its mode of action in the periphery is not. The discovery of specific cells that contain and synthesize a peptide acting by a paiacrine and/or exocrine mode of action, both in the periphery and in the central nervous system, raises many questions about somatostatin’s true physiologic activities. Is it a hormone, a paninhibin, and/or a neurotransmitter inhibitor? To answer these questions, the information we have on somatostatin’s direct and indirect inhibiting effects, relative tissue distribution, variable modes of action, and specific activities related to their location ahd natural release stimulators in the gastrointestinal tract must be carefully studied. Since its discovery [l], somatostatin’s physiologic activity in the gut and pancreas has been more clearly elucidated. Somatostatin-containing cells in the mesenteric plexus and submucosa are in proximity with cells containing other peptides with known functions. We can infer that these coexistent peptides in the gastrointestinal tract may have integrated physiologies. Likewise, the concomitant presence of cholecystokinin, neural peptide Y, calcitonin gene-related peptide, and somatostatin In the same neuron support the concept of a synergistic and/or integrated physiologic effect [2]. The objective of this article is to present an overall picture of our current knowledge of somatostatin’s physiologic activities, based on the findings of numerous studies. The data presented in the tables were derived using tissue extraction and purification methods, employing established techniques and aqueous acid extractions with enzyme inhibitors [3-51.

Influence of age and sex on basal secretion of growth hormone (GH) and on GH-induced release by porcine GH-releasing factor pGRF(1-29NH2) in growing pigs.

The aim of this study was to determine the effect of age and sex on basal secretory patterns of growth hormone (GH) and growth hormone-releasing factor (GRF) induced GH release. Eighteen pigs (9 castrated males and 9 females) were stimulated with pGRF(1-29)NH2 at 7,11,15,19 and 23 weeks of age. Blood samples were taken from each animal via jugular vein cannulae every 20 min, from 6 hr before to 5 hr after iv GRF administration at a dose of 4 micrograms/kg. GH baseline levels, amplitude of the GH peaks, area under the GH peaks and the overall mean of GH serum levels decreased (P less than .001) with age in both sexes. Age also had a marked effect on GRF-induced GH release: the amplitude of GH peaks and area under the GH peaks decreased (P less than .001) with age. The GH response to pGRF(1-29)NH2 varied considerably, depending on the timing of the episodic endogenous secretion of GH. An immediate response (less than 30 min) was observed when GRF was injected at the end of a trough period or at the beginning of a peak, but there was no immediate response when GRF was injected at the end of a peak or at the beginning of a trough period. Our results show that both endogenous GH secretion and pGRF(1-29)NH2-induced GH release declines with age, suggesting a decreased sensitivity of the somatotroph cells to GRF with age; and that the high variability of the GH response to pGRF(1-29)NH2 stimulation depends greatly on the timing of the episodic endogenous GH release, thus implying a possible episodic endogenous somatostatin secretion by the hypothalamus.

Hormonal changes following an acute stress in control and somatostatin-immunized pigs

Sixteen Yorkshire pigs (49 +/- 2 kg BW at 17 weeks) were immunized against somatostatin (SRIF; 4 males, 4 females) or its conjugated protein, bovine serum albumin (BSA; controls; 4 males, 4 females). Immunizations were done at 10, 12 and 14 weeks of age. Jugular vein cannulae were surgically inserted at 17 weeks of age. Five d later, half of each sex from the control and SRIF-immunized groups were stressed. The other half were subjected to the same stress 48 hr later. On both days, remaining animals were used as unstressed controls. The stress consisted of 5 min of snare restraint. Blood samples were collected from all pigs on both days at -20, -15, -10, -5, 0 (beginning of stress), 2, 6, 10, 15, 20, 30, 40, 60, 90, 120, 150, 180 and 240 min. Samples were radioimmunoassayed for cortisol, growth hormone (GH), prolactin (Prl), insulin, triiodothyronine (T3), thyroxine (T4) and insulin-like growth factor I (IGF-I). Mean antibody titers against SRIF (1:150 dilution) at 15 weeks were 0.49 +/- .09% and 54.5 +/- 4.9% for control and SRIF immunized pigs, respectively. Gender and immunization against SRIF had no effect on any of the variables measured (P greater than 0.05), except for T3 levels which were greater in females than in males (P less than 0.05). The stress by time of sampling interaction was significant (P less than 0.01) for all hormones measured. Cortisol values almost tripled within 15 min of stress, reaching concentrations above 100 ng/mL. Maximal increases were seen at 2 min for T4 (14%), at 6 min for T3 (36%), at 15 min for Prl (46%) and at 10 min for insulin (141%). An increase of 129% in GH concentration was present at 20 min in stressed pigs; however, an increase of 97% was also seen at 120 min in control pigs. Concentrations of IGF-I decreased (21%) by 60 min in the stressed pigs and remained depressed for up to 150 min. Stress associated with snare restraint, therefore, induces major changes in the concentrations of a series of hormones in growing pigs. On the other hand, immunization against SRIF did not alter any of the hormonal profiles measured. Since snare restraint is widely used to handle pigs during jugular puncture, any study of hormonal secretion in this species should be carried out under carefully controlled conditions in terms of blood sampling technique.

DYNAMICS OF GROWTH HORMONE RESPONSIVENESS TO GROWTH HORMONE RELEASING FACTOR IN AGING RATS PERIPHERAL AND CENTRAL INFLUENCES

The in vivo and in vitro dynamics of somatotroph responsiveness to rGRF (1-29) NH2 (rat growth hormone releasing factor) were evaluated in 2-, 4-, 8-, 12-, and 20-month-old male rats. In vivo, using pentobarbital-anesthetized animals, we observed that the rGH (rat growth hormone) responsiveness to 0.4 and 1.6 micrograms/kg rGRF started to decline at the higher dose in 12-month-old rats and was completely blunted at both rGRF doses in 20-month-old animals. In vitro, using freshly dispersed perifused pituitary cells, we also documented a decrease of rGRF-induced rGH secretion in 12- and 20-month-old rats. Moreover, as the animals aged, the rGRF-induced rGH secretion was differentially affected by the inhibiting action of somatostatin (p less than 0.001), suggesting a loss of pituitary sensitivity to somatostatin in the presence of a high concentration of rGRF. The pituitary rGH content increased until rats reached 12 months of age, but was diminished in 20-month-old rats. In contrast, the pituitary somatostatin content increased twofold in 20-month-old rats as compared with younger rats. The hypothalamic somatostatin content was highest in 8-month-old rats and only slightly diminished in 20-month-old animals. Finally, plasma insulin-like growth factor I concentrations were highest in 8-month-old rats and lowest in 20-month-old animals. Altogether, these results indicate that the physiological loss of somatotroph responsiveness associated with the process of aging starts around 12 months of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic administration of growth hormone-releasing factor increases wound strength and collagen maturation in granulation tissue

The effects of chronic administration of growth hormone-releasing factor (GRF) on wound healing were studied in rats. Cutaneous wound strength was measured by tensometry at 5, 10, and 14 days postwounding in rats implanted with a slow-release pellet which contained a compressed mixture of a fatty acid and [desamino Tyr1, D-Ala2, Ala15]hGRF(1-29)NH2 or the fatty acid alone. There was a significant increase in wound tensile strength in GRF-treated rats compared to controls at each measurement: Day 5, 130 +/- 12 vs 97 +/- 14 g; Day 10, 402 +/- 18 vs 280 +/- 11 g; Day 14, 830 +/- 17 vs 614 +/- 14 g (P less than 0.01 for each value). Granulation tissue obtained from subcutaneously implanted polyvinyl alcohol sponges encased in silicone tubing was also studied. The amount of collagen deposited in the granulation tissue was estimated by measuring the hydroxyproline (Hyp) content of sponges retrieved 5, 10, and 14 days postinsertion from GRF-treated and control rats. Hyp content (nmole/mg sponge) was similar in both treated and control animals at each measurement: Day 5, 1.7 +/- 0.2 vs 2.2 +/- 0.2; Day 10, 31.9 +/- 4.1 vs 26.7 + 0.4; and Day 14, 41.6 +/- 7.3 vs 38.5 +/- 4.4. Hyp/proline, Hyp/glycine, and glycine/total amino acid ratios, evaluated after 10 days, were also similar in both groups. Collagen from the granulation tissue of sponges retrieved after 14 days from treated and control rats was studied by electron microscopy (magnifications, 7,100 and 22,720).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of age and intake on growth hormone kinetics in dairy heifers

The effects of aging and intake on growth hormone (GH) kinetics and GH-releasing factor (GRF)-induced GH concentrations were studied in two groups of 12 Holstein heifers each (80 d, 85 kg: young; and 273 d of age, 246 kg: old). Each group was then equally subdivided into full-fed (FF) and restricted-fed (RF) subgroups. After 11 d of intake treatment, animals were infused for 3 hr with GH (1.5 mg/hr) in order to calculate GH metabolic clearance rate (MCR), secretion rate (SR) and half-life (t 1/2). Two d later, total plasma volume was determined and the following day, all heifers received a GRF challenge (5 micrograms/kg i.v.). The following values are LSM +/- SE for young-FF, young-RF, old-FF and old-RF. Rate of secretion was not affected by any treatment, averaging 1.51, 1.25, 1.34, and 1.40 +/- .23 micrograms/min. Aging increased (P < .01) MCR (186, 159, 382, and 300 +/- 21 ml/min) and increased plasma volume (P < .01), which resulted in lower basal GH concentrations. Aging also decreased (P < .01) the area under the GH response curve following GRF injection (AUC: 12442, 21114, 5155, and 6308 +/- 1776 ng.min/ml) but did not affect average GH quantity in the plasma after the GRF challenge. Feed restriction decreased (P < .05) MCR, but not enough to affect basal GH concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum growth hormone release during a 60-hour period in growing pigs

Growth hormone (GH) profiles were measured during a 60-hour period in four castrated male and five female, 20-week-old Yorkshire pigs. During this period, GH release was not affected by feeding when measured at 2, 4 and 8 hr before and after feeding time. A photoperiod of 12 hr light and 12 hr darkness produced a decrease (P less than .05) in baseline mean GH levels from 4.0 to 3.5 ng/ml during periods of darkness. This effect was observed in both genders. Females and castrated males exhibited (P greater than .05) similar baseline GH levels and identical numbers of GH peaks during feeding and photoperiod studies. However, during these periods, the amplitude of the GH peaks and areas under the GH curves were greater (P less than .05) in females. These results indicate that: 1) feeding did not influence GH secretion; 2) darkness produced a decrease in the baseline GH levels in both sexes; and 3) females secreted more GH than castrated males of the same age.

Decreased pituitary growth hormone response to growth hormone-releasing factor in cafeteria-fed rats: dietary and obesity effects.

The in vivo and in vitro growth hormone (GH) responsiveness to growth hormone-releasing factor [rGRF(1-29)NH2] was evaluated in a dietary obese rat model. Sprague-Dawley rats were divided into two groups after weaning. The control group received a semisynthetic defined diet, and the cafeteria-fed group was maintained on a mixed energy-rich palatable diet. After 2 months of diet, the cafeteria-fed rats were divided into two groups, according to their degree of weight gain compared to controls: group I: 0%; group II: 24%. After 5 months of diet, the weight increase was, respectively, in groups I and II, 12 and 41%, as compared to controls. Under pentobarbital anesthesia, rGRF(1-29)NH2 was injected intravenously in two consecutive doses of 0.8 and 4.0 micrograms/kg body weight into the control and cafeteria-fed rats. After 2 months of diet, a significant decrease of basal GH levels and GH peak response to the 4.0 micrograms/kg rGRF dose was observed in both cafeteria-fed rat groups as compared to the control group. After 5 months of diet, basal GH levels decreased in both cafeteria-fed groups. However, a significantly blunted GH response to both doses of rGRF occurred only in group II. After 5 months of diet, perifused anterior pituitary cells of control and cafeteria-fed rats were challenged with increasing concentrations of rGRF (6.25, 25 and 100 pM). The basal GH secretion was similar in all groups but the stimulated GH release in response to 25 and 100 pM GRF, expressed as peak value, was depressed in group II, compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo growth hormone responsiveness to growth hormone-releasing factor in male and female Zucker rats.

In order to determine whether there is an anomaly in the pituitary responsiveness to growth hormone (GH)-releasing factor in the genetically obese rat, we examined the in vitro and in vivo effects of rGRF(1-29)NH2 (GRF) on GH release in male and female Zucker rats. The effect of increasing GRF concentrations (1.56, 6.25, 12.5 25 and 50 pM) was first tested on GH release from freshly perifused anterior pituitary cells. In both sexes, the GH response per one pituitary equivalent to each GRF concentration tested was reduced in the obese group. However, when GH release was expressed as a percent of initial cell GH content, there was no difference between the lean and the obese groups. Furthermore, under pentobarbital anesthesia, GRF was injected intravenously at two consecutive doses of 0.8 and 4.0 microgram/kg body weight in obese and lean animals. In both sexes, the GH response to each dose of GRF tested was decreased in the obese group. Basal serum GH concentrations were similar in male and female obese rats compared to their respective lean siblings. In conclusion, this study demonstrates a decrease of the in vitro and in vivo pituitary response to GRF in the obese Zucker rat, suggesting a possible secondary defect at the pituitary level.