Interaction of kisspeptin and the somatotropic axis.

Kisspeptin, a regulator of gonadotropin-releasing hormone, has been hypothesized as an integrator of nutrition and hormones critical to metabolism and the regulation of reproduction. Growth hormone (GH) is necessary for optimal reproduction and recent evidence suggests that its secretion may be influenced by kisspeptin. The objectives of this study were to determine whether the effect of kisspeptin to stimulate GH release is due to an interaction with growth hormone-releasing hormone (GHRH) or somatostatin (SS), or an effect at the hypothalamus. Intravenous injection and infusion of kisspeptin [500 pmol/kg BW (650 ng/kg)/h × 5 h] to cows (n = 5) increased serum concentrations of luteinizing hormone (LH) but not GH. Pretreatment with kisspeptin injection and infusion in cows (n = 5) reduced the stimulatory effect of GHRH (0.05 µg/kg BW) on GH secretion. However, the magnitude of the GH response to GHRH (assessed by incremental AUC) was not affected by kisspeptin. In these same cows, administration of kisspeptin prevented the increase in GH induced by SS infusion (0.5 µg/kg BW/ h × 1.5 h) withdrawal. Peripheral administration of kisspeptin [200 and 1,000 pmol/kg BW (260 and 1,300 ng/kg)] increased serum concentrations of LH but not GH in ewes (n = 8). However, concentrations of GH were stimulated by central kisspeptin treatment [100 and 200 pmol/kg BW (130 and 260 ng/kg)] in ewes. In addition to activating the gonadotropic axis, kisspeptin can activate the somatotropic axis in ruminants. Present data support the concept of a central site of action for this effect.

Central role of the melanocortin-4 receptors in appetite regulation after endotoxin.

Melanocortin-4 receptors (MC4R) are key factors in the depression of appetite during disease. This study was designed to determine the role of agouti-related protein (AgRP) in the effect of endotoxin (lipopolysaccharide, LPS) on appetite. Sheep received an intracerebroventricular injection of either saline or AgRP (0.5 nmol/kg of BW) 1 h before intravenous injection of either saline or LPS (0.6 microg/kg of BW) at time 0 and again at 4 h. Agouti-related protein prevented the reduction in feed intake due to LPS (P < 0.05). In a second experiment, AgRP gene expression was unaffected at 3 h and increased (P < 0.01) at 6 h after LPS. Immunohistochemical evidence indicated that there was an increase in the percentage of AgRP neurons with c-Fos immunoreactive nuclei 6 h after sheep were injected with LPS (P < 0.04) and a corresponding decrease in a-melanocyte-stimulating hormone neurons coexpressing c-Fos (P < 0.001). In situ hybridization provided evidence for an increase in AgRP gene expression and a decrease in proopiomelanocortin gene expression 6 h after LPS (P < 0.05). In a final experiment, physiological elevation of orexigenic agents by short-term fasting kept feed intake at the same level as controls, in spite of the presence of LPS, similar to the effects of AgRP in Exp. 1. The AgRP inhibition of the MC4R prevents appetite inhibition in response to LPS and well after LPS inhibition of feed intake, both AgRP and a-melanocyte-stimulating hormone may change in a pattern that favors appetite increases. These studies support the notion of the MC4R as a critical component of the mechanism for appetite suppression due to endotoxin.

Long-term feed intake regulation in sheep is mediated by opioid receptors.

These experiments were conducted to determine if 1) syndyphalin-33 (SD33), a mu-opioid receptor ligand, affects feed intake; 2) SD33 effects on feed intake are mediated by actions on opioid receptors; and 3) its activity can counteract the reduction in feed intake associated with administration of bacterial endotoxin. In Exp. 1, 5 mixed-breed, castrate male sheep were housed indoors in individual pens. Animals had ad libitum access to water and concentrate feed. Saline (SAL; 0.9% NaCl) or SD33 (0.05 or 0.1 micromol/kg of BW) was injected i.v., and feed intake was determined at 2, 4, 6, 8, 24, and 48 h after the i.v. injections. Both doses of SD33 increased (at least P < 0.01) feed intake at 48 h relative to saline. In Exp. 2, SAL + SAL, SAL + SD33 (0.1 micromol/kg of BW), naloxone (NAL; 1 mg/kg of BW) + SAL, and NAL + SD33 were injected i.v. Food intake was determined as in Exp. 1. The SAL + SD33 treatment increased (P = 0.022) feed intake at 48 h relative to SAL + SAL. The NAL + SAL treatment reduced (at least P < 0.01) feed intake at 4, 6, 8, 24, and 48 h, whereas the combination of NAL and SD33 did not reduce feed intake at 24 (P = 0.969) or 48 h (P = 0.076) relative to the saline-treated sheep. In Exp. 3, sheep received 1 of 4 treatments: SAL + SAL, SAL + 0.1 micromol of SD33/kg of BW, 0.1 microg of lipopolysaccharide (LPS)/kg of BW + SAL, or LPS + SD33, and feed intake was monitored as in Exp. 1. Lipopolysaccharide suppressed cumulative feed intake for 48 h (P < 0.01) relative to saline control, but SD33 failed to reverse the reduction in feed intake during this period. These data indicate that SD33 increases feed intake in sheep after i.v. injection, and its effects are mediated via opioid receptors. However, the LPS-induced suppression in feed intake cannot be overcome by the opioid receptor ligand, SD33.

Modeling growth factor activity during proinflammatory stress: methodological considerations in assessing cytokine modulation of IGF binding proteins released by cultured bovine kidney epithelial cells.

The present research was conducted to model potential mechanisms through which IGFBPs might be affected by a key proinflammatory response initiating cytokine tumor necrosis factor (TNF-)-alpha. Madin-Darby bovine kidney epithelial (MDBK) cells, known to release IGFBPs in response to several stimuli, were grown under several conditions and challenged with forskolin (F) or recombinant TNF-alpha for 24h. Forskolin increased IGFBP-3 gene expression and media content of BP-3 protein. TNF-alpha increased basal and augmented F-mediated IGFBP-3 gene expression. However, TNF-alpha effects on the measurable media content of IGFBPs were influenced by culture conditions; in the absence of added protease inhibitors (PIs) or sufficient media albumin concentration (high BSA, 1mg/ml), the effect of TNF-alpha was to decrease (P<0.02) measurable IGFBPs. In the presence of PI and high BSA, media IGFBP-3 levels were shown to be increased by TNF-alpha consistent with the gene expression data. Changes in media IGFBP-3 protease activity were examined further to explain the observed effects of TNF-alpha on production and destruction of IGFBPs in media. When recombinant human IGFBP-3 (500 ng/ml) was added to PI-free, low BSA 100 microg/ml) media from TNF-treated MDBK cells, less than 10% of the BP-3 was recognizable by Western blot in 30 min; conversely, inclusion of High BSA and PI in media resulted in attenuation of the protease effect on the IGFBPs. The data suggest that the MDBK model of cellular response to proinflammatory stimulus is affected by culture conditions and that TNF-alpha affects media content of IGFBPs through effects on IGFBP gene expression coupled with degradation of IGFBPs via enhanced proteolytic enzyme release.

Neuropeptide Y restores appetite and alters concentrations of GH after central administration to endotoxic sheep.

The objective of this study was to determine whether neuropeptide Y (NPY) and recombinant human interleukin-1 receptor antagonist (IL-1ra) would: first, increase food intake; secondly, decrease concentrations of GH; thirdly, reduce GHRH-induced release of GH; and fourthly, reduce changes to concentrations of IGF-I in plasma during experimental endotoxemia in sheep. Six treatments were given to six castrated male sheep in a 6x6 Latin square treatment order. Osmotic mini-pumps were implanted at 0 h and a jugular vein was cannulated. Each sheep was continuously infused with saline (0.9%) or lipopolysaccharide (LPS) (20 micrograms/kg per 24 h, s.c.) at 10 microliters/h for 72 h via the osmotic mini-pumps. Blood samples (3 ml) were collected at 15-min intervals from 24 to 33 h. At 26 h, one of three treatments (artificial cerebrospinal fluid, NPY or IL-1ra) was injected i.c.v. within 30 s (0.3 microgram/kg), then infused i.c.v. from 26 to 33 h (600 microliters/h) at 0.3 microgram/kg per h. GHRH was injected i.v. (0.075 microgram/kg) at 32 h after which blood samples were collected at 5, 10, 15, 30, 45 and 60 min. Feed intake was reduced up to 50% for 48 h in LPS-treated compared with non-LPS-treated sheep. NPY restored feed intake in LPS-treated sheep and induced hyperphagia in non-LPS-treated sheep from 24 to 48 h. In contrast, IL-1ra did not affect appetite. Injection of NPY increased concentrations of GH from 26 to 27 h, while IL-1ra had no effect. Infusion of NPY suppressed GHRH-induced release of GH. However, no treatment altered pulse secretion parameters of GH. Concentrations of IGF-I were 20% higher at 72 h in LPS-treated sheep given NPY than in sheep treated with LPS alone, and this may reflect increased appetite from 24 to 48 h. We concluded that reduced appetite during endotoxemia is due to down-regulation of an NPY-mediated mechanism. Furthermore, NPY stimulates release of GH in healthy sheep, does not reduce pulse secretion parameters of GH, but does suppress GHRH-induced release of GH in endotoxic sheep. Therefore, NPY may be an important neurotransmitter linking appetite with regulation of GH during endotoxemic and healthy states in sheep.

Estradiol/progesterone implants increase food intake, reduce hyperglycemia and increase insulin resistance in endotoxic steers.

High doses of lipopolysaccharide (LPS) induce transient hyperglycemia, then chronic hypoglycemia and increased insulin resistance. In addition, appetite is reduced, while body temperature and concentrations of cortisol and tumor necrosis factor alpha (TNFalpha) are elevated. Furthermore, concentrations of GH and IGF-I are reduced in cattle. The objectives of this study were to determine whether a gonadal steroid implant (20 mg estrogen and 200 mg progesterone) given to endotoxemic steers would: (1) reduce hyperglycemia, reduce hypoglycemia, reduce insulin resistance, (2) reduce changes in concentrations of GH and IGF-I, (3) reduce inappetence and reduce concentrations of blood urea nitrogen (BUN) and non-esterified fatty acids (NEFA), and (4) reduce fever and concentrations of TNFalpha and cortisol. Holstein steers were assigned within a 2x2 factorial arrangement of treatments as follows (n=5 per group): C/C, no steroid and vehicle; S/C, steroid and vehicle; C/E, no steroid and LPS (1 microg/kg body weight (BW), i.v.); S/E, steroid and endotoxin. Steroid implants were given at 20 weeks of age (day 0) and serial blood samples (15 min) were collected on day 14 for 8 h, with vehicle or LPS injected after 2 h. Intravenous glucose tolerance tests (100 mg/kg BW) were carried out at 6 h and 24 h. Hyperglycemia was 67% lower (P<0.05) in S/E- compared with C/E-treated steers between 30 and 150 min after i.v. injection of LPS. Hypoglycemia developed after 4 h and insulin resistance was greater in S/E- compared with C/E-treated steers (P<0. 05) at 6 and 24 h. Concentrations of IGF-I were restored earlier in steroid-treated steers than in controls. Concentrations of GH were not affected by steroids, but increased 1 h after injection of LPS, then were reduced for 2 h. Appetite was greater (P<0.05) in S/E- (2.1% BW) compared with C/E-treated steers (1.1% BW) (pooled s.e.m.=0.3). Concentrations of NEFA increased after injecting LPS, but concentrations were lower (P<0.05) in S/E- compared with C/E-treated steers. LPS did not affect concentrations of BUN, but concentrations were lower in steroid-treated steers. Steroids did not affect body temperature or concentrations of TNFalpha and cortisol. In summary, gonadal steroids reduce hyperglycemia, reduce inappetence and tissue wasting, but increase insulin resistance. Furthermore, concentrations of IGF-I are restored earlier in steroid-treated than in non-steroid-treated steers injected with LPS. It is concluded that gonadal steroids reduce severity of some endocrine and metabolic parameters associated with endotoxemia. However, it is unlikely that gonadal steroids acted via anti-inflammatory and immunosuppressive actions of glucocorticoids or through reducing concentrations of cytokines.