Barbara P Steele

Interaction of Estrogen and Progesterone on Kisspeptin-10-Stimulated Luteinizing Hormone and Growth Hormone in Ovariectomized Cows

Background/Aims: Growth hormone (GH) is necessary for optimal reproductive efficiency and its secretion is influenced by sex steroids. This study was designed to determine whether kisspeptin-10 (Kp10) could stimulate GH and if gonadal steroids enhance the GH response to Kp10 in cows. Methods and Results: Intravenous injection of Kp10 at 100 or 200 pmol/kg body weight with or without treatment with estradiol cypionate and/or progesterone increased luteinizing hormone (p < 0.01) plasma concentrations. Plasma concentrations of GH were increased following Kp10 in cows treated with estradiol cypionate and/or progesterone (p < 0.05) but not in cows treated with Kp10 without gonadal steroids. Conclusions: These data suggest that reproductive steroids enhance the sensitivity of the somatotropic axis to physiologically relevant doses of Kp10, and support the possibility that Kp10 is an integrator of luteinizing hormone and GH release.

Production of a low-molecular-weight, alkaline-active, thermostable protease by a novel, spiral-shaped bacterium, Kurthia spiroforme, sp. nov.

Abstract A Gram-positive bacterium exhibiting a spiral morphology at near neutral pH was isolated from a thermal spring. Growth occurs over a temperature range of 4° to 47°C and a pH range of 7.0 to 11.5. Growth optimum is at 30°C and pH 10.5. Gas chromatographic analysis of the cellular fatty acids identified the organism as a Kurthia sp. Morphological and physiological traits differ significantly from other Kurthia sp.; therefore we have named the organism Kurthia spiroforme, sp. nov. The organism produces a unique extracellular, alkaline-active, thermostable protease with an extremely low molecular weight of 8 kilodaltons. Optimum activity of the protease is observed at pH 11.0 and 60°C. At 50°C (pH 9.0) the protease has a half-life of 41 h.

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.

A role for agouti-related protein in appetite regulation in a species with continuous nutrient delivery.

Knowledge of specific neurotransmitters as well as the pathways and mechanisms regulating appetite in ruminants that continually graze, such as sheep, is incomplete. Although fundamentally agouti-related protein (AGRP) has a similar function across species to increase food intake, the regulation of AGRP may vary across grazing and intermittent feeders. To investigate the role of orexigenic peptides in the regulation of feed intake, we first extracted messenger RNA from sheep that were fasted for 3 days, which was then used for PCR followed by cloning and sequencing to demonstrate the presence of hypothalamic AGRP expression. Ovine AGRP was closely related to the bovine, but contained sequence differences with human and mouse AGRP. Analysis of genomic DNA also revealed a similar gene structure to other published species. Secondly, using dual-labeled immunohistochemistry, we determined that there was both increased AGRP immunoreactivity and increased abundance of c-Fos immunoreactivity in AGRP neurons in the arcuate nucleus of fasted sheep. Because AGRP neurons are activated by fasting, we hypothesized that AGRP would stimulate feeding in this ruminant species. Sheep fed ad libitum were injected intracerebroventricularly with concentrations of AGRP at 0.2 and 2.0 nmol/kg. AGRP at 2.0 nmol/kg significantly increased food intake at 4, 6 and 12 h (p < 0.05). A 4th study was done to investigate the interactions of AGRP and neuropeptide Y (NPY) on food intake over a 24-hour period. Intracerebroventricular injections of either AGRP or NPY significantly increased cumulative food intake over saline controls. When AGRP and NPY were injected in combination, food intake was increased over saline controls; however, AGRP did not potentiate the effects of NPY. These results demonstrate that AGRP stimulates food intake in sheep and highlights the important differences between this species and rodent models.

Cytokine-Mediated Growth Hormone Release from Cultured Ovine Pituitary Cells

Previous studies have demonstrated that intravenous lipopolysaccharide (LPS) will increase concentrations of growth hormone (GH). One possible explanation for this may reside in the response of the pituitary to specific cytokines. This study sought to determine the effects of recombinant bovine tumor necrosis factor α (TNF), recombinant ovine (ro) interleukin-1α (IL-1α), roIL-1β, ro interleukin-2 (IL-2), and ro γ-interferon (INT) on GH release from cultured sheep pituitary cells. Sheep were sacrificed and pituitary cells cultured in DMEM with 10% fetal bovine serum for 3 days. On day 4, cells were washed and serum-free DMEM added to cells. IL-1α and IL-1β were used at 0.2, 2 and 20 ng/ml and the remaining cytokines at 2, 20 and 200 ng/ml. Neither IL-2 nor INT had effects on basal or on GH-releasing hormone (GRH)-stimulated GH release. TNF inhibited GRH-stimulated GH release (p < 0.05). Both IL-1α and IL-1β stimulated GH release from cultured pituitary cells at all doses tested (p < 0.01). Neither IL-1α nor IL-1β had an effect on GRH-stimulated GH release. IL-1 effects were inhibited by H-89 (p < 0.05; a protein kinase A inhibitor) and by nifedipine (p < 0.05; a calcium channel blocker). Both of these mechanisms are central signal transduction mechanisms mediating GRH-stimulated GH release. IL-1-stimulated GH release is partially inhibited (p < 0.05) by lipoxygenase pathway blockers. Phorbol myristate acetate downregulation of protein kinase C did not alter IL-1-stimulated GH release. IL-1β increased the content of both GH and GH mRNA in cultured sheep pituitary cells. We conclude that IL-1 produces a strong stimulus to GH release, which is mediated by calcium entry and protein kinase A activation. IL-1 also activates lipoxygenase pathways. This latter pathway as well as calcium entry were shown to mediate LPS stimulation of GH release from cultured pituitary cells. The similarity between IL-1 and LPS signal transduction suggests that LPS may activate pituitary production of IL-1 to produce the stimulus to GH. The lack of inhibitory effects of INT, TNF and IL-2 as opposed to what is seen in the rat may suggest a partial mechanism to explain the different effects of LPS on GH release between sheep and that seen in cattle and rats.

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.

Alterations in the Growth Hormone/Insulin-Like Growth Factor I Pathways in Feline GM1 Gangliosidosis1

Cats affected with feline GM1 gangliosidosis, an autosomal, recessively inherited, lysosomal enzymopathy, have progressive neurological dysfunction, premature thymic involution, stunted growth, and premature death. Although increased membrane GM1 gangliosides can result in increased apoptosis of thymocytes, there is not a direct correlation between thymocyte surface GM1 and thymic apoptosis in vivo, suggesting that other factors may be important to the pathogenesis of thymic involution in affected cats. Because GH and insulin-like growth factor I (IGF-I) are important hormonal peptides supporting thymic function and affecting growth throughout the body, particularly in the prepubescent period, several components of the GH/IGF-I pathway were compared in GM1 mutant and normal age-matched cats. GM1 mutant cat serum IGF-I concentrations were reduced significantly compared with those in normal cats by 150 days of age, and GM1 mutant cats had no peripuberal increase in serum IGF-I. Additionally, IGF-binding pro...

Kisspeptin stimulates growth hormone release by utilizing neuropeptide y pathways and is dependent on the presence of ghrelin in the ewe

&NA; Although kisspeptin is the primary stimulator of gonadotropin‐releasing hormone secretion and therefore the hypothalamic‐pituitary‐gonadal axis, recent findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Here we show that central delivery of kisspeptin causes a robust rise in plasma GH in fasted but not fed sheep. Kisspeptin‐induced GH secretion was similar in animals fasted for 24 hours and those fasted for 72 hours, suggesting that the factors involved in kisspeptin‐induced GH secretion are responsive to loss of food availability and not the result of severe negative energy balance. Pretreatment with the neuropeptide Y (NPY) Y1 receptor antagonist, BIBO 3304, blocked the effects of kisspeptin‐induced GH release, implicating NPY as an intermediary. Kisspeptin treatment induced c‐Fos in NPY and GH‐releasing hormone (GHRH) cells of the arcuate nucleus. The same kisspeptin treatment resulted in a reduction in c‐Fos in somatostatin (SS) cells in the periventricular nucleus. Finally, blockade of systemic ghrelin release or antagonism of the ghrelin receptor eliminated or reduced the ability of kisspeptin to induce GH release, suggesting the presence of ghrelin is required for kisspeptin‐induced GH release in fasted animals. Our findings support the hypothesis that during short‐term fasting, systemic ghrelin concentrations and NPY expression in the arcuate nucleus rise. This permits kisspeptin activation of NPY cells. In turn, NPY stimulates GHRH cells and inhibits SS cells, resulting in GH release. We propose a mechanism by which kisspeptin conveys reproductive and hormone status onto the somatotropic axis, resulting in alterations in GH release.