Ontogeny expression of ghrelin,neuropeptide Y and cholecystokinin in blunt snout bream,Megalobrama amblycephala

Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) all have important roles in the regulation of feeding in fish and mammals. To better understand the role of the three peptides in appetite regulation in the early developmental stages of blunt snout bream (Megalobrama amblycephala), partial cDNA sequences of ghrelin, NPY and CCK genes were cloned. And then, real‐time quantitative PCR and RT‐PCR were used to detect and quantify the mRNA expressions of these genes from zygotes to larvae of 50 days after hatching (DAH). Ghrelin, NPY and CCK were all expressed throughout the embryonic and larval development stages, and the expression levels were higher in larval stages than in embryonic stages. Ghrelin and NPY mRNA expressions were upregulated at 1, 3, 5 DAH, while CCK mRNA expression was reduced significantly at 3 DAH. The mRNA expression levels of three genes in larvae varied significantly until 30 DAH. In adult fish, all three peptides were detected to be expressed in brain and several peripheral tissues. Ghrelin mRNA was mainly expressed in the intestine, whereas NPY and CCK mRNAs were mainly expressed in the brain. Taken together, these results indicate that ghrelin, NPY and CCK may have roles in early development and participate in the regulation of feeding of larvae in blunt snout bream and will be helpful for further investigation into feed intake regulation in adults of this species.     

Inhibition of growth hormone secretagogue receptor antagonist, [D-Lys-3]-GHRP-6, in adipogenesis of ovine and rat adipocytes

Ghrelin and growth hormone secretagogues receptor (GHS‐R or ghrelin receptor) have been reported as being one of the factors of adipogenesis in adipocytes. To investigate the involvement of ghrelin and GHS‐R in adipocytes, the effect of the GHS‐R antagonist, [D‐Lys‐3]‐GHRP‐6 (His‐D‐Trp‐D‐Lys‐Trp‐D‐Phe‐Lys‐NH₂), on the process of adipogenesis in ovine and rat adipocytes was evaluated. [D‐Lys‐3]‐GHRP‐6 (10^?7 mol/L) significantly inhibited adipogenic differentiation of ovine and rat preadipocytes prepared from adipose tissues. The level of peroxisome proliferator activated receptor (PPAR)‐γ2 mRNA, an adipogenic marker, was decreased during the differentiation of adipocytes treated with [D‐Lys‐3]‐GHRP‐6 for 10 days. Ghrelin stimulated adipogenesis, also causing an increment of glycerol‐3‐phosphate dehydrogenase and upregulation of PPAR‐γ2. Furthermore, the antilipolytic effect of ghrelin was attenuated by treatment with [D‐Lys‐3]‐GHRP‐6 in both types of isolated adipocytes. Overall, the results of the present study highlight that GHS‐R in adipogenesis can be blocked by treatment with [D‐Lys‐3]‐GHRP‐6.     

胚胎及新生期蛋鸡和肉鸡垂体生长激素基因表达的比较研究

本文采用Northern杂交分析法研究了14胚龄、18胚龄、1日龄、5日龄及10日龄肉鸡和蛋鸡垂体生长激素的基因表达，同时采用放射免疫分析法（RIA）测定了垂体内生长激素的含量。结果表明：14胚龄时在肉鸡和蛋鸡垂体总RNA中均未出生长激素mRNA(GH mRNA)。从18胚龄开始可检测出一条0.8kb的GHmRNA，并且垂体GHmRNA水平的发育性变化在品种间呈现不同的规律：蛋鸡从18胚龄到10日龄垂体GHmRNA水平不断升高，日龄间差异显著（P＜0.05）。肉鸡从18胚龄到出壳1日龄，GHmRNA水平有较大幅度的升高，但从1日龄至10日龄维持在1日龄时的水平。18胚龄肉鸡垂体GH mRNA水平显著高于蛋鸡（P＜0.05），并在1日龄和5日龄均维持在较高水平，与生长速度呈正相关；而10日龄时垂体HGmRNA水平的品种差异发生逆转，蛋鸡GHmRNA水平反而高于肉鸡。垂体HG含量的发育性变化趋势与垂体HGmRNA水平相一致，10日龄时蛋鸡垂体GH含量显著高于肉鸡（P＜0.05），与生长速度呈相反的趋。     

Role for des-acyl ghrelin in the responsiveness of plasma hormones and metabolites to ghrelin in Holstein steers

Gastric-derived peptide hormone ghrelin is known for its potent growth hormone (GH) stimulatory effects. The acyl-modification on N-terminal Ser(3) residue is reported to be important to stimulate the ghrelin receptor, GH secretagogue-receptor type1a (GHS-R1a). However, major portion of circulating ghrelin lacks in acylation, and some biological properties of des-acyl ghrelin have been reported in monogastric animals. In the present study, the responsiveness of plasma hormones and metabolites to ghrelin in steers was characterized, and role for des-acyl ghrelin in these changes was investigated. The repeated intravenous administrations of bovine ghrelin (1.0 microg/kg BW) every 2h for 8h to Holstein steers significantly increased the plasma acylated ghrelin, total ghrelin, GH, insulin and NEFA levels. The GH responses in peak values and area under the curves (AUCs) were attenuated by repeated injections of ghrelin, however, the responses of plasma total ghrelin were similar. Plasma insulin AUC decreased after fourth injection of ghrelin while plasma NEFA AUCs gradually increased by repeated injections of ghrelin. Pretreatment of des-acyl ghrelin (10.0 microg/kg BW) 5 min prior to the single injection of ghrelin (1.0 microg/kg BW) did not affect the ghrelin-induced hormonal changes. Moreover, the responses of plasma GH to bovine and porcine ghrelin, which differ in C-terminal amino acid residues, were similar in calves. These data show that (1) GH release was attenuated by repeated administration of ghrelin, (2) ghrelin regulates glucose and fatty acid metabolism probably via different pathway, and (3) des-acyl ghrelin is unlikely the antagonist for ghrelin to induce endocrine effects in Holstein steers.     

Regulation of growth hormone expression by thyrotropin-releasing hormone through the pituitary-specific transcription factor Pit-1 in chicken pituitary

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone beta- (TSHbeta) encoding genes. Pit-1 has been identified in several mammalian and avian species. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and it stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully developed a competitive RT-PCR for the detection of Pit-1 expression in the chicken pituitary, that was sensitive enough to detect picogram levels of Pit-1 mRNA. Applying this method, the effect of TRH injections on Pit-1 mRNA expression was determined in the pituitary of chick embryos and growing chicks. In both 18-day-old embryos and 10-day-old male chicks the Pit-1 mRNA expression was significantly increased following TRH injection, thereby indicating that the stimulatory effects of TRH on several pituitary hormones is mediated via its effect on Pit-1 expression. Therefore, a semi-quantitative RT-PCR method was used to detect possible changes in GH levels. TRH affected the GH mRNA levels at both developmental stages. These results, combined with the data on Pit-1 mRNA expression, indicate that Pit-1 has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH.     

Gonadotrophin assay of cephalic and caudal lobes of anterior pituitary from growing turkeys

Pituitaries from male and female turkeys reared under natural daylight were collected every 2 weeks. Each pituitary was divided into the cephalic and caudal lobe and then homogenised and frozen until assayed. Gonadotrophic activity was determined by the P32 uptake of day‐old chick testes. Significant differences were found associated with the age of the birds, but not with either the sex or the lobe of the gland, nor any of the possible interactions. The peak activity was reached when the birds were 22 weeks of age, and a slight but non‐significant decrease took place until 34 weeks of age when the experiment was terminated.

A significant correlation (P≤0.01) between the amount of naturally occurring daylight and anterior pituitary gonadotrophin concentration of 0.86 was found. The interpretation of this correlation is discussed.     

IGFBP-3基因在两个品种鸡组织中的表达及其与生长性状的相关性分析

为研究IGFBP-3基因表达与鸡生长性状的相关性,以生长速度差异较大的花山麻鸡和清远麻鸡两个黄羽肉鸡品种为研究素材,用实时荧光定量PCR法检测胚胎期和出雏后两个品种鸡胸肌和肝脏中IGFBP-3基因的表达规律,并将其与体重、胸肌重和肝脏重进行相关性分析。结果表明,在9胚龄时两个品种鸡胸肌和肝脏中均可检测到IGFBP-3基因表达,同一胚龄或日龄品种内组织间比较,胚胎期两个品种鸡胸肌IGFBP-3 mRNA表达量均高于肝脏,出雏后肝脏IGFBP-3 mRNA表达量迅速上升,胸肌IGFBP-3 mRNA表达量下降,两个品种鸡肝脏IGFBP-3 mRNA表达量均极显著高于胸肌（P<0.01）;组织中IGFBP-3 mRNA表达量与组织重量和体重相关研究表明,两个品种鸡肝脏IGFBP-3 mRNA表达量与其胸肌重、肝脏重和体重呈显著或极显著正相关（P<0.05;P<0.01）,胸肌IGFBP-3 mRNA表达量与其胸肌重、肝脏重和体重均呈极显著负相关（P<0.01）。研究结果揭示,IGFBP-3 mRNA在鸡中的表达具有品种、年龄和组织特异性,出雏前肝脏并不是鸡产生IGFBP-3的主要器官,出雏后鸡IGFBP-3可能主要由肝脏合成,肝脏中IGFBP-3 mRNA水平差异是导致两个品种鸡出雏后体重和胸肌重差异的重要因素。     

Changes in ghrelin levels of plasma and proventriculus and ghrelin mRNA of proventriculus in fasted and refed layer chicks

This is a test-report of ghrelin levels in plasma and proventriculus, the glandular portion of the avian stomach, by using a specific radioimmunoassay for acylated ghrelin, as well as the expression of the ghrelin gene in the proventriculus after a 12-h fasting period followed by a 6-h feeding period with 6-day-old layer chicks. After fasting, the plasma ghrelin levels increased from 21.3 ± 4.5 to 32.9 ± 5.0 fmol/ml, but once refed it returned to the control value. After fasting, the ghrelin mRNA and the peptide levels in the proventriculus increased, and ghrelin mRNA levels remained high but once refed the ghrelin content returned to the control level. Furthermore, in order to examine the effect of increased circulating ghrelin on food intake, a bolus intravenous injection of 500 pmol of chicken ghrelin was given to 8-day-old chicks. The ghrelin injection did not cause any significant changes in food intake. These results indicate that the levels of ghrelin and its mRNA with layer chicks are altered according to the feeding state and this in a similar manner as has been observed in mammals. Unlike in mammals, an increase in circulating ghrelin does not cause the promotion of food intake in chicks.     

The release of growth hormone (GH): relation to the thyrotropic- and corticotropic axis in the chicken

In the chicken and other avian species, the secretion of GH is under a dual stimulatory and inhibitory control of hypothalamic hypophysiotropic factors. Additionally, the thyrotropin-releasing hormone (TRH), contrary to the mammalian situation, is also somatotropic and equally important in releasing GH in chick embryos and juvenile chicks compared to the (mammalian) growth hormone-releasing hormone (GHRH) itself. Consequently, the negative feedback loop for GH release not only involves the insulin-like growth factor IGF-I but also thyroid hormones. In adult chickens, TRH does no longer have a clear thyrotropic activity, whereas its somatotropic activity depends on the feeding status of the animal. In addition, as in mammals, the secretion of GH and glucocorticoids is stimulated by ghrelin, a novel peptide predominantly synthesized in the gastrointestinal tract. Two chicken isoforms of the ghrelin receptor have been identified, both of which are highly expressed in the hypothalamus and pituitary, suggesting that a stimulatory effect may be directed at these levels. GH and glucocorticoids control the peripheral thyroid hormone function by down-regulating the hepatic type III deiodinating enzyme (D3) in embryos (GH and glucocorticoids) and in juvenile and adult chickens (GH). Moreover, glucocorticoids help to regulate T3-homeostasis in the brain during embryogenesis by stimulating the type II deiodinase (D2) expression. This way not only a multifactorial release mechanism exists for GH but also a functional entanglement of activities between the somatotropic-, thyrotropic- and corticotropic axis.     

Regulation of pituitary somatotroph differentiation by hormones of peripheral endocrine glands

Anterior pituitary somatotroph differentiation occurs during chick embryonic and rat fetal development. A number of findings support the hypothesis that differentiation of these growth hormone (GH) producing cells in the chick and the rat is regulated by adrenal glucocorticoids and thyroid hormones. Somatotroph differentiation can be induced in cultures of chick embryonic and rat fetal pituitary cells with adrenal glucocorticoids and this effect can be modulated by concomitant treatment with thyroid hormones. Plasma levels of thyroid hormones, corticosterone and adrenocorticotropic hormone increase during development, consistent with the ontogeny of somatotrophs. Treatment of chick embryos or rat fetuses with glucocorticoids in vivo induces premature somatotroph differentiation, indicating that the adrenal gland, and ultimately anterior pituitary corticotrophs, may function to regulate pituitary GH cell differentiation during development. Administration of thyroid hormones in vivo also increases somatotrophs prematurely, and administration of the thyroid hormone synthesis inhibitor methimazole inhibits somatotroph differentiation in vivo, suggesting that endogenous thyroid hormone synthesis contributes to normal somatotroph differentiation. Our working model for the regulation of somatotroph differentiation during normal development includes modulation by elements of the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-thyroid axes. Additional research is reviewed defining the mechanism of action for these peripheral hormones in induction of pituitary GH gene expression during development.     

Hepatic lipolysis in broiler chickens with different fat deposition during embryonic development

The aim of this study was to explore the hepatic lipolysis in broiler chickens with different fat deposition during embryonic development. The mRNA expression of CPT-1 (carmitine palmtoyltransferase-1), PPARα (peroxisome proliferator-activated receptor alpha) and LPL (lipoprotein lipase) genes were determined using Real time RT-PCR. The start of incubation was called day 1 (E1) and after hatching called day 1 (H1). On incubation days 9 (E9), 14 (E14) and 19 (E19) as well as at hatching (H1), samples of liver were collected. Blood samples were obtained during days 14 (E14) and 19 (E19) of embryonic development and at hatching. This study showed that serum TG (triglycerol) decreased and TC (total cholesterol) and NEFA (non-estered fatty acid) increased during embryonic development. The expression of CPT-1, PPARα and LPL genes exhibited different developmental changes. For example, little LPL gene was expressed at hatching and PPARα gene expression peaked before hatching. However, CPT-1 gene exhibited no significance during the embryonic development. Our results showed that expression of these genes in Arbor Acres (AA) broilers was significantly higher than that in San Huang (SH) broilers. Therefore, this study suggested that hepatic lipolysis in broiler chickens exhibited developmental changes during embryogenesis and breed difference which may be one of the factors in the fat deposition difference between fat line and lean line broilers during embryonic development.     

The chicken pituitary-specific transcription factor PIT-1 is involved in the hypothalamic regulation of pituitary hormones

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone-beta- (TSHbeta) encoding genes. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.     

Ghrelin对雌激素诱导小鼠胸腺萎缩过程中细胞因子及凋亡相关蛋白基因表达的影响

应用光镜和实时定量PCR技术研究了Ghrelin对雌激素诱导小鼠胸腺萎缩过程中胸腺形态学以及部分细胞因子和凋亡相关蛋白基因表达的影响。结果显示,注射Ghrelin后,雌激素诱导的小鼠萎缩胸腺在形态学上基本恢复到正常水平,胸腺中IL-6、TGF-[31、Caspase3、FADDmRNA含量显著降低,Caspase9、FasLmRNA含量略为下降,而IL-7、Bcl-2mRNA含量有所上升。结果表明,Ghrelin可能通过促进胸腺上皮细胞的增殖以及阻断Caspase级联程序和Fas／FasL凋亡信号通路抑制胸腺细胞的凋亡,从而逆转雌激素诱导的小鼠胸腺萎缩。