Effects of nesfatin-1 on food intake and LH secretion in prepubertal gilts and genomic association of the porcine NUCB2 gene with growth traits.

Nesfatin-1, a product of the nucleobindin 2 (NUCB2) gene, purportedly plays important roles in whole-body energy homeostasis. Experiments were conducted to determine how NUCB2 expression in fat depots may be controlled in the pig and to test the hypothesis that nesfatin-1 regulates appetite and LH secretion in the gilt. Prepubertal gilts were used to study expression of NUCB2 in fat and the effects of intracerebroventricular (i.c.v.) injection of nesfatin-1 on food intake and pituitary hormone secretion. Growing pigs (gilts and barrows at 22 wk of age, n = 1,145) or sexually mature gilts (n = 439) were used to test association of SNP in the NUCB2 gene with growth traits. The expression of NUCB2 was similar for subcutaneous fat compared with perirenal fat. An i.c.v. injection of the melanocortin-4 receptor agonist [Nle4, d-Phe7]-α-melanocyte-stimulating hormone did not alter expression of NUCB2 mRNA in the hypothalamus but reduced (P = 0.056) NUCB2 mRNA expression in subcutaneous fat. Short-term (7 d) submaintenance feeding reduced (P < 0.05) BW and did not alter expression of mRNA for NUCB2, visfatin, or leptin but increased (P < 0.05) expression of adiponectin mRNA in fat. Central injection of nesfatin-1 suppressed (P < 0.001) feed intake. Secretion of LH was greater (P < 0.01) after i.c.v. injection of nesfatin-1 than after saline. Single nucleotide polymorphisms in the porcine NUCB2 gene were not associated with adiposity of growing pigs or age at puberty in gilts but were associated (P < 0.05) with BW at puberty. These data indicate that NUCB2 is expressed in fat depots of the pig and that the level of expression is sensitive to stimulation of appetite-regulating pathways in the hypothalamus. It is confirmed herein that nesfatin-1 can regulate appetite in the pig and affect the gonadotropic axis of the prepubertal pig. Association of SNP in the porcine NUCB2 gene with BW at puberty suggests that regulation of appetite by nesfatin-1 in the pig affects growth, which may have important consequences for adult phenotypes.

Expression of genes for interleukins, neuropeptides, growth hormone receptor, and leptin receptor in adipose tissue from growing broiler chickens.

In this study, total RNA was collected from abdominal adipose tissue samples obtained from 10 broiler chickens at 3, 4, 5, and 6 wk of age and prepared for quantitative real-time PCR analysis. Quantitative real-time PCR analysis was used to examine the influence of age on the expression of the adipose tissue genes for IL-1ß, -6, -10, -15, -18; brain-derived neurotropic factor; ciliary neurotropic factor; interferon γ, neuropeptide Y receptor Y1; neuropeptide Y; nucleobindin 2; growth hormone receptor; leptin receptor; and visfatin. Between 3 and 6 wk of age, leptin receptor expression decreased (P=0.013) with age, whereas expression of IL-15 (P=0.015) and growth hormone receptor (P=0.002) increased. Furthermore, IL-18 (P<0.001) and visfatin (P=0.007) expression increased between 4 and 6 wk of age. This is a unique exhibition of age-related changes in cytokine gene expression in chicken adipose tissue. Future studies are needed to elucidate the role of adipose tissue cytokines in growth and, possibly, disease resistance.

Effects of organic selenium supplementation on growth performance, carcass measurements, tissue selenium concentrations, characteristics of reproductive organs, and testis gene expression profiles in boars.

The objective was to compare growth and physiological responses in boars fed diets supplemented with organic or inorganic sources of Se. At weaning, crossbred boars (n = 117; 8.3 kg of BW) were placed in nursery pens (3 boars/pen) and assigned within BW blocks to receive on an ad libitum basis 1 of 3 dietary treatments: I) basal diets with no supplemental Se (controls), II) basal diets supplemented with 0.3 mg/kg of organic Se, and, III) basal diets supplemented with 0.3 mg/kg of sodium selenite (13 pens/dietary treatment). Average daily gain (470 g/d), ADFI (896 g/d), and G:F (0.54) were similar among groups. Blood Se concentrations were greater (P < 0.01) for boars consuming organic Se (107.5 ± 4.8 µg/L) or sodium selenite (114.7 ± 4.8 µg/L) compared with controls (28.4 ± 4.8 µg/L). Intact pens of boars (11 pens/dietary treatment) were moved to a grow-finish barn and continued to receive appropriate diets on an ad libitum basis. Average daily gain (1,045 g/d) and ADFI (2,716 g/d) were similar among groups. Gain:feed was affected by treatment (P = 0.02) and was greater (P < 0.06) for boars fed organic Se (0.378 ± 0.004) compared with boars fed sodium selenite (0.368 ± 0.004) or controls (0.363 ± 0.004). Blood Se concentrations were greater (P < 0.01) in grow-finish boars consuming organic Se (198.9 ± 5.5 µg/L) than boars consuming sodium selenite (171.4 ± 5.4 µg/L) or controls (26.7 ± 5.4 µg/L). Treatment did not affect (P > 0.15) HCW, dressing percent, carcass length, LM area, standardized fat-free lean, lean percentage, backfat thickness, visual color, firmness, marbling, or Minolta loin color scores. Selenium supplementation did not affect (P > 0.17) testis or accessory sex gland sizes. Concentrations of Se in loin, liver, kidney, testis, cauda epididymis, and accessory sex glands were greatest (P < 0.01) in boars receiving organic Se, intermediate in boars receiving sodum selenite, and least in control boars. Microarray analysis of testis gene expression did not detect differences (P > 0.05) due to dietary treatment. Testis gene expression of glutathione peroxidase 4, as determined using quantitative PCR, was increased (P < 0.01) in boars fed organic Se compared with those fed sodium selenite. In summary, dietary supplementation of boars with organic Se failed to alter ADG or ADFI but enhanced G:F during grow-finish. More research is needed to discern the mechanism by which organic Se improves feed efficiency in boars.

Gene expression profiling in developing pig adipose tissue: non-secreted regulatory proteins.

The expression of many genes encoding secreted and non-secreted factors have been studied in human and rodent adipose tissue with cDNA microarrays, but few such studies in adipose tissue from growing pigs have been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) samples from gilts at 90, 150 and 210 days (n = 5/age). Dye-labeled cDNA probes were hybridized to custom microarrays (70-mer oligonucleotides) representing about 600 pig genes involved in growth and reproduction. Gene expression intensity ratios changed little with age for 100 transcription factors, nuclear receptors, enzymes and other regulatory proteins in OSQ and MSQ from pigs between 90 and 210 days of age. However, the relative expression of 13 genes distinguished OSQ and MSQ depots in growing pigs. The expression of several genes were influenced by age including an increase in CCND3, HSF1 and PTGR1 expression in MSQ and a decrease in UCP2 and REA (prohibitin-2) expression in OSQ. These studies demonstrate for the first time the expression of several key regulatory genes in pig adipose tissue. Simple linear regression analysis showed that leptin gene expression was associated with expression of some of these regulatory genes. Negative associations between expression of some regulatory factors and leptin gene expression indicated that local leptin may decrease or antagonize adipogenesis.

Luteinizing hormone secretion as influenced by age and estradiol in the prepubertal gilt.

The aim of this study was to determine if there is an age related reduction in the sensitivity of the negative feedback action of 17ß-estradiol (estradiol) on luteinizing hormone (LH) secretion in the prepubertal gilt. Ovariectomized gilts at 90 (n=12), 150 (n=11) or 210 (n=12) days of age received estradiol benzoate (EB) osmotic pump implants 6/group and the remaining animals received vehicle control (C) implants except for 150-day C (n=5) on Day 0. On Day 10 blood samples were collected every 15 min for 8h and serum LH and estradiol concentrations were measured. Serum estradiol concentrations averaged 5 ± 1, 5 ± 1 and 7 ± 2 pg/ml for the 90-, 150- and 210-day-old gilts implanted with estradiol, respectively, whereas, serum estradiol concentrations was undetectable in C gilts. Mean serum LH concentrations, basal LH concentrations and serum LH pulse amplitude were less in EB-treated gilts at all ages compared to control animals. In contrast, LH pulse frequency initially was less in EB-treated gilts but subsequently increased (P<0.04) with age (from 0.8 ± 0.2 at 90 days to 5.2 ± 0.2/8h at 210 days), and at 210 days of age the pulse frequency was similar to C gilts. These results demonstrate an age related reduction in the sensitivity to the negative feedback action of estradiol on LH secretion and support the idea that the gilt conforms to the gonadostat hypothesis.

Adipose tissue and the reproductive axis: biological aspects.

The discovery of leptin has clearly demonstrated a relationship between body fat and the neuroendocrine axis since leptin influences appetite and the reproductive axis. Since adipose tissue is a primary source of leptin, adipose tissue is no longer considered as simply a depot to store fat. Recent findings demonstrate that numerous other genes, i.e. neuropeptides, interleukins and other cytokines and biologically active substances such as leptin and insulin-like growth factors I and II, are also produced by adipose tissue, which could influence appetite and the reproductive axis. Targets of leptin in the hypothalamus include neuropeptide Y, proopiomelanocortin and kisspeptin. Transsynaptic connection of hypothalamic neurons to porcine adipose tissue may result in a direct influence of the hypothalamus on adipose tissue function. Nutritional signals such as leptin are detected by the central nervous system and translated by the neuroendocrine system into signals which ultimately regulates luteinizing hormone secretion. Furthermore, leptin directly affects gonadotropin-releasing hormone release from the hypothalamus, luteinizing hormone from the pituitary gland and ovarian follicular steroidogenesis. Although leptin is identified as a putative signal that links metabolic status and neuroendocrine control of reproduction, other adipocyte protein products may play key roles in regulating the reproductive axisin the pig.

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

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

Energy metabolism and leptin: effects on neuroendocrine regulation of reproduction in the gilt and sow.

It is well established that reproductive function is metabolically gated. However, the mechanisms whereby energy stores and metabolic cues influence appetite, energy homeostasis and fertility are yet to be completely understood. Adipose tissue is no longer considered as only a depot to store excess energy. Recent findings have identified numerous genes, several neurotrophic factors, interleukins, insulin-like growth factor binding protein-5, ciliary neurotrophic factor and neuropeptide Y (NPY) as being expressed by adipose tissue during pubertal development. These studies demonstrated for the first time the expression of several major adipokines or cytokines in pig adipose tissue which may influence local and central metabolism and growth. Leptin appears to be the primary metabolic signal and is part of the adipose tissue-hypothalamic regulatory loop in the control of appetite, energy homeostasis and luteinizing hormone (LH) secretion. Leptin's actions on appetite regulation are mediated by inhibition of hypothalamic NPY and stimulation of proopiomelanocortin. Its effects on gonadotropin-releasing hormone (GnRH)/LH secretion are mediated by NPY and kisspeptin. Thus, leptin appears to be an important link between metabolic status, the neuroendocrine axis and subsequent fertility in the gilt and sow.

Patterns of gene expression in pig adipose tissue: insulin-like growth factor system proteins, neuropeptide Y (NPY), NPY receptors, neurotrophic factors and other secreted factors.

Although cDNA microarray studies have examined gene expression in human and rodent adipose tissue, only one microarray study of adipose tissue from growing pigs has been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) from gilts at 90, 150, and 210 d (n=5 age(-1)). Dye labeled cDNA probes were hybridized to custom porcine microarrays (70-mer oligonucleotides). Gene expression of insulin-like growth factor binding proteins (IGFBPs), hormones, growth factors, neuropeptide Y (NPY) receptors (NPYRs) and other receptors in OSQ and MSQ changed little with age in growing pigs. Distinct patterns of relative gene expression were evident within NPYR and IGFBP family members in adipose tissue from growing pigs. Relative gene expression levels of NPY2R, NPY4R and angiopoietin 2 (ANG-2) distinguished OSQ and MSQ depots in growing pigs. We demonstrated, for the first time, the expression of IGFBP-7, IGFBP-5, NPY1R, NPY2R, NPY, connective tissue growth factor (CTGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes in pig adipose tissue with microarray and RT-PCR assays. Furthermore, adipose tissue CTGF gene expression was upregulated while NPY and NPY2R gene expression were significantly down regulated by age. These studies demonstrate that expression of neuropeptides and neurotrophic factors in pig adipose tissue may be involved in regulation of leptin secretion. Many other regulatory factors were not influenced by age in growing pigs but may be influenced by location or depot.

Hypothalamic neurons innervating fat tissue in the pig express leptin receptor immunoreactivity.

While leptin receptors have been found in both the autonomic ganglion neurons and the hypothalamic nuclei, studies dealing with the projections from the central nervous system to the adipose tissue have been conducted mainly in laboratory animals. Therefore, the purpose of our study was to establish whether hypothalamic neurons are transsynaptically connected to adipose tissue depots in the pig, and if these neurons express leptin receptor immunoreactivity. Pseudorabies virus (PRV; Bartha's K strain) was introduced in perirenal or subcutaneous adipose tissue depots in domestic pigs. On day 9, animals were euthanized and hypothalami were collected and processed immunohistochemically with primary antisera against PRV and leptin receptor (OBR). PRV-labeled neurons were localized in paraventricular nucleus, supraoptic nucleus and arcuate nucleus following injections in both the perirenal and the subcutaneous adipose tissue depots. Ventromedial nucleus, dorsomedial nucleus and preoptic area-labeled neurons were observed after injection of the PRV into the perirenal adipose tissue, while in the lateral hypothalamic area-labeled neurons projected only to the subcutaneous adipose tissue. The majority of the PRV-labeled neurons simultaneously expressed OBR-immunoreactivity. Our results provide the morphological data on multisynaptic projections from hypothalamus to the fat tissue in the pig and demonstrate that these neurons, located in areas involved in reproductive processes and feeding behavior, may regulate fat tissue metabolism.

Patterns of gene expression in pig adipose tissue: transforming growth factors, interferons, interleukins, and apolipoproteins.

Although cDNA microarray studies have indicated the expression of unique and unexpected genes and their products in human and rodent adipose tissue, cDNA microarray studies of adipose tissue from growing pigs have not been reported. Total RNA was collected at slaughter from outer s.c. adipose tissue (OSQ), middle s.c. adipose tissue (MSQ), ovary, uterus, hypothalamus, and pituitary tissue samples from gilts at 90, 150, and 210 d (n = 5/age). Dye-labeled cDNA probes were hybridized to custom microarrays (70 mer oligonucleotides) representing approximately 600 pig genes involved in growth and reproduction. Expression intensity ratios revealed little change in expression of 27 cytokines and 4 apolipoproteins with age in OSQ and MSQ from pigs at 90, 150, and 210 d of age. Distinct patterns of relative gene expression were evident within apolipoproteins, IL, interferons, and transforming growth factor beta family members in adipose tissue from growing pigs (90-, 150-, and 210-d-old pigs). Patterns of gene expression within apolipoproteins, IL, interferons, and transforming growth factor beta family members distinguished OSQ and MSQ depots in growing pigs. We also demonstrated, for the first time, the expression of several major cytokine and apolipoprotein genes in pig adipose tissue, including small inducible cytokine A5 (RANTES), IL-1B, IL-1A, IL-12A, IL-1 receptor antagonist, and apolipoproteins A1 and E with microarray and reverse transcription-PCR assays or reverse transcription-PCR assays alone. These studies demonstrate that expression of major cytokine and apolipoprotein genes in pig adipose tissue are not influenced by age in growing pigs but may be influenced by location or depot.

Gene expression in the brain-pituitary adipose tissue axis and luteinising hormone secretion during pubertal development in the gilt.

The occurrence of puberty in the female is due to the interplay of central and peripheral mechanisms in which the hypothalamic-pituitary-ovarian axis regulates growth and gonadal function, as well as adipocyte hormone secretion. Hypothalamic GnRH mRNA expression increased at 3.5 months of age and declined by 6 months of age. Concomitant with the age related reduction in the oestrogen negative feedback on LH secretion was a decline in hypothalamic oestrogen receptor-alpha (ERalpha) expression and increased expression of repressor of ER activity gene (REA) at 210 days of age. Hypothalamic proopiomelanocortin expression increased at 6 months of age followed by increased expression of progesterone receptor (PR) membrane compliment-1 and steroid membrane binding protein gene at 210 days of age. This represents development of the endogenous opioid peptide-progesterone dependent LH inhibitory pathway. Adipose tissue leptin and insulin like growth factor-I (IGF-I) gene expression increased with age and adiposity. Pituitary transcription factors, steroidogenic factor 1 (SF1) and Lhx3, and LHbeta and FSHbeta gene expression increased with age. These results identify key hypothalamic and pituitary genes associated with changes in LH secretion and growth during pubertal development and adipose tissue genes and secreted proteins related to maturation of the neuroendocrine axis and puberty.

Secreted proteins and genes in fetal and neonatal pig adipose tissue and stromal-vascular cells.

Although microarray and proteomic studies have indicated the expression of unique and unexpected genes and their products in human and rodent adipose tissue, similar studies of meat animal adipose tissue have not been reported. Thus, total RNA was isolated from stromal-vascular (S-V) cell cultures (n = 4; 2 arrays; 2 cultures/array) from 90-d (79% of gestation) fetuses and adipose tissue from 105-d (92% of gestation) fetuses (n = 2) and neonatal (5-d-old) pigs (n = 2). Duplicate adipose tissue microarrays (n = 4) represented RNA samples from a pig and a fetus. Dye-labeled cDNA probes were hybridized to custom microarrays (70-mer oligonucleotides) representing more than 600 pig genes involved in growth and reproduction. Microarray studies showed significant expression of 40 genes encoding for known adipose tissue secreted proteins in fetal S-V cell cultures and adipose tissue. Expression of 10 genes encoding secreted proteins not known to be expressed by adipose tissue was also observed in neonatal adipose tissue and fetal S-V cell cultures. Additionally, the agouti gene was detected by reverse transcription-PCR in pig S-V cultures and adipose tissue. Proteomic analysis of adipose tissue and fetal and young pig S-V cell culture-conditioned media identified multiple secreted proteins including heparin-like epidermal growth factor-like growth factor and several apolipoproteins. Another adipose tissue secreted protein, plasminogen activator inhibitor-1, was identified by ELISA in S-V cell culture media. A group of 20 adipose tissue secreted proteins were detected or identified using the gene microarray and the proteomic and protein assay approaches including apolipoprotein-A1, apolipoprotein-E, relaxin, brain-derived neurotrophic factor, and IGF binding protein-5. These studies demonstrate, for the first time, the expression of several major secreted proteins in pig adipose tissue that may influence local and central metabolism and growth.

Neuropeptide Y modulates growth hormone but not luteinizing hormone secretion from prepuberal gilt anterior pituitary cells in culture.

Pituitary cells, from seven 160- to 170-day-old pigs, were studied in primary culture to determine the affects NPY on LH and GH secretion at the level of the pituitary. On day 4 of culture, medium was discarded, plates were rinsed twice with serum-free medium and cells were cultured in 1 ml fresh medium without serum and challenged individually with 10(-10), 10(-8) or 10(-6) M [Ala(15)]-h growth hormone-releasing factor-(1-29)NH(2) (GRF); 10(-9), 10(-8) or 10(-7) M GnRH or 10(-9), 10(-8), 10(-7) or 10(-6) M NPY individually or in combinations with 10(-9) or 10(-8) M GnRH or 10(-8) or 10(-6)M GRF. Cells were exposed to treatment for 4 h at which time medium was harvested and quantified for LH and GH. Basal LH secretion (control; n = 7 pituitaries) was 12 +/- 6 ng/well. Relative to control at 4 h, 10(-9), 10(-8) and 10(-7) M GnRH increased (P < 0.01) LH secretion by 169, 176 and 197%, respectively. Neuropeptide-Y did not alter (P > 0.4) basal LH secretion nor 10(-8) M GnRH-induced increase in LH secretion but 10(-9) M GnRH-stimulated LH secretion was reduced by NPY and was not different from control or GnRH alone. Basal GH secretion (control; n = 7 pituitaries) was 56 +/- 12 ng/well. Relative to control at 4 h, 10(-10), 10(-8) and 10(-6) M GRF increased GH secretion by 111%, 125% (P < 0.01) and 150% (P < 0.01), respectively. Only 10(-6) M (134%) and 10(-7) M (125%) NPY increased (P < 0.04) basal GH secretion. Addition of 10(-9), 10(-8) and 10(-7) M NPY in combination with 10(-8) M GRF suppressed (P < 0.04) GRF-stimulated GH secretion. However, 10(-9) M NPY enhanced (P < 0.06) the GH response to 10(-6) M GRF. These results demonstrate that NPY may directly modulate GH secretion at the level of the pituitary gland.

The control of adenohypophysial hormone secretion by amino acids and peptides in swine.

Several different amino acids and peptides control secretion of adenohypophysial hormones and this control may be indirect, via the modulation of hypothalamic hormone secretion. Indeed, classical hypothalamic hormones (e.g., gonadotropin-releasing hormone [GnRH], growth hormone-releasing hormone [GHRH], somatostatin, etc.) may be released into the hypothalamo-hypophysial portal vasculature, travel to the adenohypophysis and there stimulate or inhibit secretion of hormones. Alternatively, some amino acids and peptides exert direct stimulatory or inhibitory effects on the adenohypophysis, thereby impacting hormone secretion. In swine, the most extensively studied modulators of adenohypophysial hormone secretion are the excitatory amino acids (ExAA), namely glutamate and aspartate, and the endogenous opioid peptides (EOP). In general, excitatory amino acids stimulate release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and prolactin (PRL). Secretion of adenohypophysial hormones induced by ExAA is primarily, but perhaps not exclusively, a consequence of action at the central nervous system. By acting primarily at the level of the central nervous system, EOP inhibit LH secretion, stimulate GH release and depending on the animal model studied, exert either stimulatory or inhibitory influences on PRL secretion. However, the EOP also inhibited LH release by direct action on the adenohypophysis. More recently, peptides such as neuropeptide-Y (NPY), orexin-B, ghrelin, galanin, and substance P have been evaluated for possible roles in controlling adenohypophysial hormone secretion in swine. For example, NPY, orexin-B, and ghrelin increased basal GH secretion and modulated the GH response to GHRH, at least in part, by direct action on the adenohypophysis. Secretion of LH was stimulated by orexin-B, galanin, and substance P from porcine pituitary cells in vitro. Because the ExAA and various peptides modulate secretion of adenohypophysial hormones, these compounds may play an important role in regulating swine growth and reproduction.

Leptin: a metabolic signal affecting central regulation of reproduction in the pig.

The discovery of the obesity gene and its product, leptin, it is now possible to examine the relationship between body fat and the neuroendocrine axis. A minimum percentage of body fat may be linked to onset of puberty and weaning-to-estrus interval in the pig. Adipose tissue is no longer considered as only a depot to store excess energy in the form of fat. Recent findings demonstrate that numerous genes, i.e., relaxin, interleukins and other cytokines and biologically active substances such as leptin, insulin-like growth factor-I (IGF-I), IGF-II and Agouti protein are produced by porcine adipose tissue, which could have a profound effect on appetite and the reproductive axis. Hypothalamic neurons are transsynaptically connected to porcine adipose tissue and may regulate adipose tissue function. In the pig nutritional signals such as leptin are detected by the central nervous system (CNS) and translated by the neuroendocrine system into signals, which regulate appetite, hypothalamic gonadotropin-releasing hormone (GnRH) release and subsequent luteinizing hormone (LH) secretion. Furthermore, leptin directly affects LH secretion from the pituitary gland independent of CNS input. Changes in body weight or nutritional status are characterized by altered adipocyte function a reduction in adipose tissue leptin expression, serum leptin concentrations and a concurrent decrease in LH secretion. During pubertal development serum leptin levels, hypothalamic leptin receptor mRNA and estrogen-induced leptin gene expression in fat increased with age and adiposity in the pig and this occurred at the time of expected puberty. In the lactating sow serum and milk leptin concentrations were positively correlated with backfat thickness and level of dietary energy fed during gestation as well as feed consumption. Although, these results identify leptin as a putative signal that links metabolic status and neuroendocrine control of reproduction, other adipocyte protein products may play an important role in regulating the reproductive axis in the pig.

Orexin-B modulates luteinizing hormone and growth hormone secretion from porcine pituitary cells in culture.

To test the hypothesis that orexin-B acts directly on the anterior pituitary to regulate LH and growth hormone (GH) secretion, anterior pituitary cells from prepuberal gilts were studied in primary culture. On day 4 of culture, 10(5) cells/well were challenged with 0.1, 10 or 1000 nM GnRH; 10, 100 or 1000 nM [Ala15]-hGRF-(1-29)NH2 or 0.1, 1, 10 or 100 nM, orexin-B individually or in combinations with 0.1 and 1000 nM GnRH or 10 and 1000 nM GRF. Secreted LH and GH were measured at 4 h after treatment. Basal LH and GH secretion (control; n = 6 pigs) was 183 +/- 18 and 108 +/- 4.8 ng/well, respectively. Relative to control at 4 h, all doses of GnRH and GRF increased (P < 0.0001) LH and GH secretion, respectively. All doses of orexin-B increased (P < 0.01) LH secretion, except for the 0.1 nM dose. Basal GH secretion was unaffected by orexin-B. Addition of 1, 10 or 100 nM orexin-B in combinations with 0.1 nM GnRH increased (P < 0.001) LH secretion compared to GnRH alone. Only 0.1 nM (P = 0.06) and 100 nM (P < 0.001) orexin-B in combinations with 1000 nM GnRH increased LH secretion compared to GnRH alone. All doses of orexin-B in combination with 1000 nM GRF suppressed (P < 0.0001) GH secretion compare to GRF alone, while only 0.1 nM orexin-B in combination with 10 nM GRF suppressed (P < 0.01) GH secretion compared to GRF. These results indicate that orexin may directly modulate LH and GH secretion at the level of the pituitary gland.

Role of leptin in the regulation of gonadotropin secretion in farm animals.

The recently discovered protein, leptin, which is secreted by fat cells, has been implicated in regulation of feed intake or energy balance and the neuroendocrine axis in rodents, humans and large domestic animals. Leptin was first identified as the gene product found to be deficient in the obese (ob/ob) mouse. Administration of leptin to ob/ob mice restored reproduction as well as reducing feed intake and causing weight loss. The leptin receptor (LR) which has been cloned and is a member of the class 1 cytokine family of receptors, is found in the brain and pituitary of all species studied to date. Neuropeptide Y has been proposed as the primary mediator of leptin action in the hypothalamus to regulate luteinizing hormone (LH) and growth hormone (GH) secretion. In vitro studies using both hypothalamic explants and pituitary cell culture provided evidence that supports a direct action of leptin at the level of brain and pituitary gland in the pig, but only the pituitary in cattle. Central administration of leptin increased LH secretion in the fasted cow and ewe, but not in control fed animals, indicating that metabolic state is an important factor in modulating the hypothalamic-pituitary response to leptin. Changing serum leptin concentrations and leptin mRNA expression were associated with onset of puberty in heifers and gilts. Thus, leptin appears to be an important link between metabolic status and the neuroendocrine axis.

The role of melanocortin-3 and -4 receptor in regulating appetite, energy homeostasis and neuroendocrine function in the pig.

A recently discovered class of receptors, melanocortin-3 and -4 receptor (MC3/4-R), are located within the brain and modulate feed intake in rodents. Stimulation of the receptor (agonist) inhibits feed intake whereas blockade (antagonist) of the receptor increases intake. Our knowledge of factors regulating voluntary feed intake in humans and domestic animals is very limited. i.c.v. administration of an MC3/4-R agonist, NDP-MSH, suppressed (P<0.05) feed intake compared with controls at 12, 24, 48 and 72 h after treatment in growing pigs. Fed pigs were more responsive to the MC3/4-R agonist then fasted animals. However, i.c.v. treatment with MC3/4-R antagonist, SHU9119, failed to stimulate intake. The failure of MC3/4-R antagonist to stimulate feed intake suggests involvement of other brain hormone(s) which antagonize the action of SHU9119 at the MC3/4-R, blocking its stimulatory effect on intake. Treatment with NDP-MSH or SHU9119, across a wide dose range, failed to affect LH and GH secretion, except for the 10 micro g dose of NDP-MSH, which exhibited both a stimulatory and an inhibitory effect on GH secretion in fasted animals. Treatment with agouti-related peptide, a natural brain hormone that blocks the MC3/4R, failed to stimulate feed intake. These results do not support the idea that endogenous melanocortin pays a critical role in regulating feed intake and pituitary hormone secretion in the pig. SHU9119 blocked the NDP-MSH-induced increase in cAMP in HEK293 cells expressing the porcine MC4-R sequence without the missense mutation. The EC(50) and IC(50) values were similar to the human MC4-R, confirming that SHU9119 is a pig MC4-R antagonist. However, pigs were heterozygous for an MC4-R gene missense mutation. It is possible that the MC4-R mutation alters function and this may explain the failure to demonstrate MC3/4-R involvement in modulating feeding behavior and LH and GH secretion in the pig.

Role of leptin in modulating the hypothalamic-pituitary axis and luteinizing hormone secretion in the prepuberal gilt.

Three experiments (EXP) were conducted to test the hypothesis that leptin modulates LH, GnRH, and neuropeptide Y (NPY) secretion. In EXP I, prepuberal gilts received intracerebroventricular (i.c.v.) leptin injections and blood samples were collected. In EXP II, anterior pituitary cells from prepuberal gilts in primary culture were challenged with 10(-14), 10(-13), 10(-12), 10(-11), 10(-10), 10(-9), 10(-8), 10(-7), or 10(-6) M leptin individually or in combinations with 10(-10), 10(-9), and 10(-8) M GnRH. In EXP III, hypothalamic-preoptic area (HYP-POA) explants were placed in perfusion system and exposed to 0 (n=5), 10(-12) M (n=4), 10(-10) M (n=4), 10(-8) M (n=4), or 10(-6) M (n=5) human recombinant leptin (LEP) for 30 min. In EXP I, serum LH concentrations were unaffected by leptin treatment. In EXP II, all doses of leptin increased LH secretion except for 10(-12) and 10(-7) M. Only 10(-7), or 10(-13) M leptin in combination with 10(-8) or 10(-9) M GnRH, respectively, suppressed LH secretion. In EXP III, prior to leptin, media GnRH concentrations were similar across treatments. Media GnRH concentrations increased after 10(-12), 10(-10), and 10(-8) M leptin compared to control. Leptin treatment failed to influence NPY secretion across treatments. These results indicate that components of the neuroendocrine axis that regulate GnRH and LH secretion are functional and leptin sensitive before the onset of puberty. Other neural peptides in addition to NPY may mediate the acute effects of leptin on the GnRH-LH system and lastly, the inability of i.c.v. leptin treatment to increase LH secretion may in part be related to stage of sexual maturation and associated change in negative feedback action of estradiol on LH secretion.