F.C. Buonomo

Determination of insulin-like growth factor 1 (IGF1) and IGF binding protein levels in swine

A heterologous radioimmunoassay system was validated for the determination of IGF1 concentrations in swine sera. Parallelism, accuracy and response to physiological stimuli were obtained following the incubation of serum samples with 1M glycine-glycine HC1 buffer at a pH of 3.5 +/- 0.2 for 24 hours at 37C. Following acidification and neutralization, circulating IGF1 concentrations were significantly (P less than .05) reduced in hypophysectomized swine and elevated in swine injected with porcine growth hormone (pGH) when compared to IGF1 levels in control hogs. IGF binding protein levels were also increased following GH administration and reduced by hypophysectomy. In addition, circulating IGF1 concentrations were significantly (P less than .05) correlated with body size in three types of swine which differ in growth rate and mature body weight. These data suggest that IGF1 is involved in the regulation of swine growth in vivo and that its physiologic regulation is similar to that in humans.

Ontogeny of growth hormone (GH), insulin-like growth factors (IGF-I and IGF-II) and IGF binding protein-2 (IGFBP-2) in genetically lean and obese swine.

Serum GH, IGF-I, IGF-II and IGFBP-2 concentrations were determined by radioimmunoassay in swine of genetic lines which were selected for high (obese) and low (lean) backfat. Blood samples were collected at birth, before and after nursing, at 1 and 3 days of age and at weekly or fortnightly intervals until 30 weeks of age. Overall, GH, IGF-I, IGF-II and IGFBP-2 were highest at birth and declined during the first week of postnatal life. An age-by-line interaction was apparent for GH and IGF-I during the early neonatal period with levels being higher in the lean line than the obese line at 1 day of age and similar at 1 week of age. At 3 to 5 weeks of age there was an elevation in GH which was greater in lean than obese pigs. IGFBP-2 concentration patterns were characterized by a nadir at 5 to 7 weeks of age and a decline from an apex at 8 weeks of age in both lines. IGF-II declined steadily from birth until about 10 weeks of age. A subsequent increase in IGF-II was then observed between 12 and 22 weeks, which was greater in the obese line and in male pigs but not apparent in lean females. At birth, pigs which had not nursed had higher GH and IGFBP-2 and lower IGF-I and IGF-II concentrations. The effect of nursing on IGF-I was significantly influenced by line.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal changes in steroids, prolactin and growth hormone in pregnant and pseudopregnant gilts during mammogenesis and lactogenesis

Abstract Estrogen, progesterone and prolactin are involved in control of mammary growth in all species studied to date. The primary hormonal change temporally related to mammary development in swine is the estrogen rise which begins on day 60 of pregnancy. However, hormonal regulation of mammary development in swine is poorly understood. Plasma was obtained at 15-day intervals (days 15–112) throughout pregnancy, day of parturition and day 4 of lactation to determine relationships between hormonal profiles and mammary development in swine. Fetal plasma was also obtained from day 60 until parturition. Additional gilts were made pseudopregnant and sampled on days 60, 90 and 112. Plasma samples were assayed for estrogens, progesterone, prolactin and growth hormone. Maternal profiles of estrone- and estradiol-sulfate, increased to 2.6 and 0.6 ng/ml, repectively on day 30 of pregnancy, with return to basal concentrations by day 45. Periparturient concentrations of estrone and estradiol did reach 13 and 2.6 ng/ml, respectively. Free estrogen concentrations (estrone and estradiol) did not change significantly at day 30, but were elevated to 6 and 0.7 pg/ml at parturition. Progesterone concentration fluctuated only slightly from a mean concentration of 25 ng/ml until the preparturient decline, resulting in concentrations of 150 ng/ml). With the exception of estrone, hormonal concentrations in estrogen-treated pseudopregnant gilts did not differ from pregnant counterparts, demonstrating that pseudopregnant gilts are an excellent in vivo model to study estrogen effects on mammary development in swine.

The neurophysiological regulation of growth hormone secretion

With the advent of genetic engineering, the importance of GH in the regulation of growth and metabolism in domestic species has been clearly demonstrated. Ample evidence of an integral role for GH in the processes of growth and lactation exists in dairy cattle (1,2), sheep (3), beef cattle (4) and swine (5). For example, circulating GH levels are high during the period of rapid growth in several species including cattle (6), swine (7) and poultry (8). Endogenous GH secretion is primarily controlled by the central nervous system (CNS) via two specific hypothalamic neurohormones, growth hormone-releasing factor (GRF) and somatostatin (SRIF), an inhibitor of GH release. The secretion of GRF and SRIF is governed by a host of neuropeptides and neurotransmitters which provide a functional link between higher CNS centers and hypophysiotropic neurons. This review will focus on the CNS regulation of GH secretion and circulating factors which feedback to either stimulate or inhibit its release.

Postnatal changes in circulating concentrations of growth hormone, somatomedin C and thyroid hormones in pigs

Plasma concentrations of growth hormone (GH), somatomedin (SmC), thyroxine (T4) and triiodothyronine (T3) were determined from birth to post weaning in pigs (Yorkshires). Plasma samples were obtained from the smallest, median and largest piglet from 11 litters. No differences in the circulating concentrations of any of the hormones were observed between piglets of different sizes. However, there were changes in circulating concentrations of hormones during postnatal development. Plasma concentrations of GH decreased between 2 and 8 to 10 days of age. A progressive increase in the circulating concentrations of SmC was observed with concentrations rising (3.83-fold between 2 days and 40 days of age). Plasma concentrations of T4 and T3 were maximal at 23 and 16 days of age, respectively.

Growth hormone releasing factors and secretion of growth hormone in sheep, calves and pigs

Abstract Human pancreatic growth hormone releasing factors (hpGRF) (1–40) and (1–44) were administered iv in sheep, pigs and calves to determine their effectiveness in stimulating GH release in these species. Both peptides produced a rapid increase in plasma GH concentration in all three species at dose levels ranging from .0065 to .65 nmol/kg. Moreover, there was no difference in the GH-secretory response observed between hpGRF(1–44)NH 2 and (1–40)OH in sheep. Sheep also responded to hpGRF(1–40)NH 2 and (1–40)OH as well as [his 1 ]- and [tyr 1 ]-hpGRF(1–40)NH 2 in a similar manner. Rat hypothalamic GRF was less effective than [his 1 ]-hpGRF(1–40), while the response to bGRF was not significantly different from hpGRF(1–40) in stimulating GH secretion in sheep. Although all three species responded to hpGRF, the elevation in plasma GH levels above baseline were greater after hpGRF injection in sheep than in pigs or calves. Subcutaneous injection of hpGRF in sheep was an effective mode of administration of the peptide, although the effect was not as long-lasting as that after iv injections and higher doses were required to stimulate GH secretion.

Suppression of somatotroph function induced by growth hormone treatment in neonatal pigs.

The effect of recombinant porcine growth hormone (pGH) treatment on pituitary function was evaluated in young pigs. Piglets received intraperitoneal recombinant pGH implants (0.5 mg/d sustained release) or vehicle implants beginning at 3 d of age. Ten piglets were sacrificed at 4 and 6 wk of age (five piglets/treatment group) for the collection of pituitary glands, blood, and liver tissue. Blood samples also were drawn at 3 and 12 d of age. Serum concentrations of GH, prolactin (PRL), thyroid-stimulating hormone (TSH), insulin-like growth factor-1 (IGF-1) and IGF-2 were evaluated. Levels of IGF-1 and IGF-2 mRNA were determined in liver samples. Treatment with GH increased circulating levels of GH and IGF-1 (P < 0.01), but not PRL, TSH, or IGF-2. Hepatic IGF-1, but not IGF-2, mRNA levels were increased by pGH (P < 0.001). Cultured pituitary cells from each animal were challenged with 0.1, 1, and 10 nM GH-releasing hormone (GHRH); 2 nM 8-Br-cAMP; or 100 nM phorbol myristate acetate. The release of GH from cultured pituitary cells was stimulated by all secretagogues (P < 0.001). The secretion of GH, but not PRL or TSH, in culture was inhibited by previous in vivo GH treatment (P < 0.001). Similarly, cellular GH, but not PRL or TSH, content was lower in the GH-implant group (P = 0.005). Cell cultures from 6-wk-old piglets secreted more GH, but not PRL or TSH, than cultures from 4-wk-old piglets (P < 0.05). Likewise, cellular GH, but not PRL or TSH, content was greatest in cultures from 6-wk-old animals (P = 0.002). Piglet growth was not affected by exogenous GH treatment (P = 0.67). These results demonstrate that exogenous pGH treatment selectively down-regulates somatotroph function in young pigs.

Effects of insulin-like growth factor I (IGF-I) on growth hormone-releasing factor (GRF) and thyrotropin-releasing hormone (TRH) stimulation of growth hormone (GH) secretion in the domestic fowl (Gallus domesticus)

Recent studies in mammalian species indicate that IGF-I may act as a negative feedback inhibitor of GH release through alteration of pituitary secretion or sensitivity to hypothalamic regulatory factors. Although avian GH secretion appears to be regulated by the differential release of hypothalamic inhibitory (somatostatin) and stimulatory (GRF and TRH) factors, feedback effects of IGF-I on in vivo GH release in birds have not been investigated. To study the effects of elevated IGF-I concentration on GRF- and TRH-stimulated GH secretion, 4-week-old chickens received an intravenous injection of recombinant human IGF-I either 15 min prior to (6 micrograms, study 1), or simultaneous with (10 micrograms, study 2). GRF (hGRF44NH2, 5 micrograms/kg) or TRH (0.5 microgram/kg) administration. Radioimmunoassay analysis of plasma collected prior to and following peptide treatment indicated that circulating IGF-I concentrations were elevated 83.9, 60.6, 77.9, and 88.8% at the time of TRH and GRF administration in studies 1 and 2, respectively. Peak GH concentrations (mean of +5- and +15-min samples) subsequent to TRH injection were significantly (P less than 0.01) depressed 45.1 and 48.2% in IGF-I-treated as compared with control chicks in the first and second studies, respectively. GRF-stimulated GH secretion was significantly (P less than 0.01) decreased by IGF-I administration in study 2 (41.3%) but not in study 1. An estimated half-life for IGF-I in the chicken is less than 15 min based on the disappearance rate of the elevation produced by exogenous IGF-I injections. Thus, IGF-I exerts a negative feedback effect on pituitary hormone secretion in avian as well as mammalian species.

Determination of circulating levels of insulin-like growth factor II (IGF-II) in swine

A heterologous radioimmunoassay system was developed for the determination of circulating IGF-II concentrations in swine. The assay utilized a monoclonal antibody against human IGF-II (Amano Intl. Ez, VA) and bovine IGF-II (Monsanto Co., MO) as the cold standard and iodinated ligand. Serial dilutions of acid-ethanol extracted normal swine sera resulted in a curve which was parallel to the bovine IGF-II standard curve. Recovery of unlabeled standard added to extracted swine sera was 101%. Neither IGF-I nor insulin were capable of cross-reacting in this assay at levels up to 100-fold excess. Using this assay, serum IGF-II levels were determined to be significantly lower when subnormal growth hormone (GH) levels existed such as in hypophysectomized swine. However, in contrast to serum IGF-I concentrations, supranormal levels of porcine GH (pGH) did not elevate serum IGF-II concentrations after 13 wk of treatment in 25 kg hogs (initial body wt). In addition, serum IGF-II levels were reduced in fasted swine, despite a significant increase in circulating GH concentrations. Thus, although normal concentrations of GH are required for maintenance of physiological levels of IGF-II in swine, the mechanism for stimulation of IGF-II secretion is less GH-dependent than IGF-I.

Amphiphilic growth hormone releasing factor (GRF) analogs: Peptide design and biological activity in vivo

The first twenty-nine amino acids of human Growth Hormone Releasing Factor (hGRF) possess a distinct amphiphilic character. This is seen as twisted hydrophobic and hydrophilic bands in the helical net projection. Four amidated analogs were designed by optimizing amphiphilic and helical potentials of the native sequence. These designed analogs, with up to eight-amino acid changes, were tested in sheep via intravenous injection. The growth hormone-stimulating activities of the analogs were significantly higher when compared to bovine Growth Hormone Releasing Factor (bGRF44-NH2). This suggests that the amphiphilic conformation of GRF(1-29) is important to the receptor.