ghrelin对生殖系统的调节作用

ghrelin是新近发现的一个含有28个氨基酸残基的多肽,是生长激素促分泌素受体(GHS-R)的天然配体,除具有调节GH分泌和能量平衡的功能之外,尚有其他许多功能。近年来体外或体内试验研究表明,ghrelin对生殖激素如LH、PRL具有一定的调节作用;另外,ghrelin及其受体系统广泛存在于生殖系统中。提示这一新发现的激素可能对生殖系统具有重要的调节作用。文章就ghrelin对生殖系统调节作用的研究进展加以综述。     

Ghrelin生物学功能的研究进展

Ghrelin是一种在大鼠和人胃内新发现的生长激素促分泌素受体(GHS-R)的内源性配基,有28个氨基酸,起促生长激素释放作用。当Ghrelin与位于垂体和下丘脑的GHS-R结合后,产生一系列生物学效应。本文从生长激素释放、机体生长发育、食欲和采食量、能量代谢、胃酸分泌等方面综述了Ghrelin的生物学功能,并初步探讨其在畜牧业上的应用前景。     

禽类Ghrelin研究进展

Ghrelin是从小鼠的胃中提纯并鉴定了的一种促生长激素分泌受体的特定内源性配体,是一种新的脑肠肽,由胃黏膜分泌,能影响哺乳动物的摄食、饮水和消化道的活动以及动物体内其他激素的分泌、从而影响其生长发育,关于Ghrelin在禽类的报道较少.为了更全面的了解禽类Ghrelin的研究作进展,为开展禽类Ghrelin的研究提供...     

Ghrelin:一个重要脑肠肽生物学效应的研究进展

Ghrelin是第一个被发现的生长激素促分泌素受体(GHS-R)的内源性配体。这个脑肠肽在调节生长激素释放、增进食欲、调节能量代谢等方面发挥重要作用,他还具有其他重要的生物学功能。加强Ghrelin在营养生理学上的研究具有重要的理论意义和广阔的应用前景。     

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.     

驯鹿生长素Ghrelin cDNA的克隆

从驯鹿皱胃组织中提取总RNA,根据已发表的驯鹿生长素Ghrelin基因序列设计并合成引物,通过反转录-聚合酶链式反应(RT-PCR)进行cDNA扩增,获得了300 bp的片段,重组到pBlueselect T载体,经限制性内切酶谱分析和DNA序列测定分析,确认PCR产物为Ghrelin cDNA,为进一步研究Chrelin在驯鹿体内的分布及营养因素等对其基因表达的影响奠定基础。     

Ghrelin and truncated ghrelin variant plasmid vectors administration into skeletal muscle augments long-term growth in rats

Ghrelin is an acylated peptide recently identified as an endogenous ligand for the growth hormone (GH) secretagogues (GHSs) receptor (GHS-R) and is involved in a novel system for regulating GH release. To study the biological activities of ghrelin using plasmid vector administration, we constructed myogenic expression vectors containing the full length cDNA of swine ghrelin-28 (pGEM-wt-sGhln) and truncated variant (pGEM-tmt-sGhln) consisting of the first seven residues of ghrelin (including Ser3 substituted with Trp3) with addition of a basic amino acid, Lys (K) at the C-terminus. After intramuscular injection of pGEM-wt-sGhln and pGEM-tmt-sGhln, RT-PCR analysis demonstrated that the ectopic expressions of ghrelin and its variant were observed 30 days post-injection. The level of GH increased in rat serum, and was significantly higher than that of the control group 20 days post-injection with pGEM-tmt-sGhln (P < 0.05). Administration of 150 microg of pGEM-wt-sGhln and pGEM-tmt-sGhln enhanced growth in rats over 30 days and great stimulatory responses were observed at day 10 and 20 post-injection respectively, whose body weight gains were on average 15% (P < 0.05) and 21% P < 0.033 significantly heavier than controls. These results suggested that skeletal muscle might have the potential to perform post-translational acylation for ghrelin, and short ghrelin variant might have the biological effects as wild type ghrelin.     

Oxyntomodulin increases the concentrations of insulin and glucose in plasma but does not affect ghrelin secretion in Holstein cattle under normal physiological conditions

Ghrelin, the natural ligand of the growth hormone secretagogue receptor (GHS-R1a), has been shown to stimulate growth hormone (GH) secretion. Regulation of ghrelin secretion in ruminants is not well studied. We investigated the effects of oxyntomodulin (OXM) and secretin on the secretions of ghrelin, insulin, glucagon, glucose, and nonesterified fatty acids (NEFA) in pre-ruminants (5 wk old) and ruminants (10 wk old) under normal physiological (feeding) conditions. Eight male Holstein calves (pre-ruminants: 52 ± 1 kg body weight [BW]; and ruminants: 85 ± 1 kg BW) were injected intravenously with 30 μg of OXM/kg BW, 50 μg of secretin/kg BW, and vehicle (0.1% bovine serum albumin [BSA] in saline as a control) in random order. Blood samples were collected, and plasma hormones and metabolites were analyzed using a double-antibody radioimmunoassay system and commercially available kits, respectively. We found that OXM increased the concentrations of insulin and glucose but did not affect the concentrations of ghrelin in both pre-ruminants and ruminants and that there was no effect of secretin on the concentrations of ghrelin, insulin, and glucose in these calves. We also investigated the dose-response effects of OXM on the secretion of insulin and glucose in 8 Holstein steers (401 ± 1 d old, 398 ± 10 kg BW). We found that OXM increased the concentrations of insulin and glucose even at physiological plasma concentrations, with a minimum effective dose of 0.4 μg/kg for the promotion of glucose secretion and 2 μg/kg for the stimulation of insulin secretion. These findings suggest that OXM takes part in glucose metabolism in ruminants.     

Circulating ghrelin and leptin concentrations and growth hormone secretagogue receptor abundance in liver, muscle, and adipose tissue of beef cattle exhibiting differences in composition of gain

Data from species other than cattle indicate that ghrelin and GH secretagogue receptor (GHS-R) could play a key role in fat deposition, energy homeostasis, or glucose metabolism by directly affecting liver and adipose tissue metabolism. Beef steers (n = 72) were used to test the hypothesis that plasma ghrelin and leptin concentrations and abundance of the GHS-R in liver, muscle, and adipose tissues differ in steers exhibiting differences in composition of gain. At trial initiation (d 0), 8 steers were slaughtered for initial carcass composition. The remaining 64 steers were stratified by BW, allotted to pen, and treatment was assigned randomly to pen. Steers were not implanted with anabolic steroids. Treatments were 1) a low-energy (LE) diet fed during the growing period (0 to 111 d) followed by a high-energy (HE) diet during the finishing period (112 to 209 d; LE-HE) or 2) the HE diet for the duration of the trial (1 to 209 d; HE-HE). Eight steers per treatment were slaughtered on d 88, 111, 160, and 209. Carcass ninth, tenth, and eleventh rib sections were dissected for chemical composition and regression equations were developed to predict compositional gain. Liver, muscle, and subcutaneous adipose tissues were frozen in liquid nitrogen for subsequent Western blotting for GHS-R. Replicate blood samples collected before each slaughter were assayed for ghrelin and leptin concentrations. When compared at a common compositional fat end-point, the rate of carcass fat accretion (g·kg of shrunk BW(-1)) was greater (P < 0.001) in HE-HE steers whereas the rate of carcass protein accretion (g·kg of shrunk BW(-1)) was less (P < 0.001) compared with LE-HE steers. When compared at a common compositional fat end-point, plasma leptin, ghrelin, and insulin concentrations were greater (P < 0.05) for HE-HE compared with LE-HE steers. Abundance of the GHS-R, to which ghrelin binds, increased over time in liver and adipose tissue but did not differ as a result of treatment. Plasma ghrelin concentrations were increased for cattle continuously fed the HE diet as they became increasingly fatter; however, abundance of the GHS-R in liver, muscle, and subcutaneous adipose tissue was not different between treatment groups. The role of ghrelin in cattle metabolism warrants further investigation as it could have a significant effect on composition of BW gain, feed efficiency, and metabolic disorders such as ketosis and fatty liver.     

禽类与哺乳类Ghrelin结构及功能研究进展

Ghrelin主要是由胃分泌的一种脑肠肽激素,它是生长激素促分泌素受体(GHSR)的内源性配基。目前,对哺乳动物Ghrelin研究相对较多,主要集中在Ghrelin结构及其对激素分泌、摄食、能量代谢、胃肠功能、生殖与免疫的调节作用,而对禽类Ghrelin结构和功能研究相对较少且稍显滞后。本文就禽类与哺乳类Ghrelin结构及功能进行比较研究作一综述,为进一步研究禽类Ghrelin的功能提供参考。     

Effects of ghrelin and its analogues on chicken ovarian granulosa cells

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1-28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1-18 and 1-5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P(4)), testosterone (T), estradiol (E(2)) and arginine-vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1-28, GHRP6 and ghrelin 1-18, but not by ghrelin 1-5. Expression of bcl-2 was suppressed by addition of ghrelin 1-28, GHRP6 and ghrelin 1-5, but promoted by ghrelin 1-18. The occurrence of PCNA was reduced by ghrelin 1-28, GHRP6, ghrelin 1-18 and ghrelin 1-5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1-28, GHRP6 and ghrelin 1-18, but not ghrelin 1-5. The accumulation of PKA decreased after treatment with ghrelin 1-28 and increased after treatment with GHRP6 and ghrelin 1-18 but not ghrelin 1-5. Secretion of P(4) by ovarian follicular fragments was decreased after addition of ghrelin 1-28 or ghrelin 1-5 but stimulated by GHRP6 and ghrelin 1-18. Testosterone secretion was inhibited by ghrelins 1-28 and 1-18, but not by GHRP6 or ghrelin 1-5. Estradiol secretion was reduced after treatment with ghrelin 1-28 but stimulated by ghrelins 1-18 and 1-5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1-28, GHRP6 and ghrelin 1-18, but inhibited by ghrelin 1-5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions-apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5-18 may be responsible for the major effects of ghrelin on the avian ovary.     

Purification and characterization of feline ghrelin and its possible role

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser3, has been isolated from rat and human stomach as an endogenous ligand for the growth hormone secretagogue receptor. Here, we purified feline ghrelin and examined its possible physiological role in cats. The major active form of feline ghrelin is a 28-amino acid peptide octanoylated (C8:0) at Ser3; except for one amino acid residue replacement, this structure is identical to those of rat and human ghrelins. However, much structural divergence in peptide length and fatty acid modification was observed in feline ghrelin: peptides consisting of 27 or 26 amino acids lacking Gln14 and/or Arg28 were found, and the third serine residue was modified by octanoic acid (C8:0), decanoic acid (10:0), or unsaturated fatty acids (C8:1, C10:1 and C10:2). In agreement with the structural divergence, two kinds of cDNA with different lengths were isolated. Administration of synthetic rat ghrelin increased plasma growth hormone levels in cats, with a potency similar to that in rat or human. Plasma levels of ghrelin in cats increased approximately 2.5-fold after fasting. The present study indicates the existence of structural divergence in feline ghrelin and suggests that, as in other animals, ghrelin may play important roles in GH release and feeding in cats.     

Expression of the Orexigenic Peptide Ghrelin and the Type 1a Growth Hormone Secretagogue Receptor in Sheep Oocytes and Pre-implantation Embryos Produced In Vitro

Recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we provide evidence that both ghrelin and its receptor GHSR-1a (the type 1a growth hormone secretagogue receptor) are expressed at both mRNA and protein levels in sheep oocytes and pre-implantation embryos produced in vitro . Real-time RT-PCR experiments confirmed that ghrelin mRNA levels varied depending on developmental stage, with the highest expression in metaphase II (MII) oocytes, higher expression at the 2-cell stage, and minimal expression in germinal vesicle (GV) oocytes, 4- and 8-cell stages, and in the blastocyst. The levels of GHSR-1a mRNA decreased from GV to MII, increased immediately at the 2-cell stage and then remained stable until the blastocyst stage. Ghrelin protein was detected mainly in the cytoplasm close to the plasma membrane in both inner cell mass and trophectoderm cells, while GHSR-1a protein was most abundant in the plasma membrane. In conclusion, the presence of the ghrelin signalling system within the sheep oocytes and pre-implantation embryos opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.     

Molecular characterization and expression profile of ghrelin gene during different reproductive phases in buffalo (Bubalus bubalis)

Ghrelin, a novel motilin-related endogenous ligand for growth hormone secretagouge receptor, is implicated in various biological functions, including regulation of female reproduction. But the presence of ghrelin and its role in reproductive functions in buffalo, a species with poor reproductive efficiency, is not known. In the present study full-length ghrelin cDNA was isolated from bubaline abomasum, which encodes the entire prepropeptide of 116 amino acids. The deduced amino acid sequence of ghrelin of buffalo showed >95% and 31% identity with that of ruminants (cattle, sheep, and goat) and humans, respectively. Analysis of synonymous and nonsynonymous nucleotide substitutions in the coding region of ghrelin indicated that these sequences of different species have been under purifying selection. The 3995-bp amplicon of ghrelin gene consisting of 4 exons and 3 introns was cloned with genomic DNA from buffalo. Further, ghrelin expression was determined by quantitative real-time PCR, in situ hybridization, and immunohistochemistry in bubaline endometrial tissues at different stages of the estrous cycle and early pregnancy. Our results indicated the persistent expression of ghrelin mRNA and protein in the endometrium during stage I (day 3–5), stage II (day 6–15), and stage III (day 16–21) of the estrous cycle and also during early (∼day 30–40) pregnancy. Immunohistochemistry and quantitative real-time PCR experiments indicated the relatively higher expression of ghrelin in the endometrium during stage II (day 6–15) of the estrous cycle and early pregnancy than during stage I (day 3–5) and stage III (day 16–21) of the estrous cycle, but no statistically significant difference in ghrelin expression was observed among stages. To conclude, the results of the present study indicate the persistent expression of ghrelin in the uterine endometrium throughout the estrous cycle and in early pregnancy which might be helpful in determining its role in buffalo reproduction.     

Performance and serum parameters of calves (Bos taurus) subject to milk restriction associated with supplementation with 2-hydroxy-4-(methylthio)butanoic acid

Our aim with this study was to evaluate the consumption, performance, quantitative characteristics of carcasses, biochemical profile, plasma levels of ghrelin and leptin, expression of the receptor for ghrelin (GHS-R1a) in the hypothalamus and duodenum, and the number of goblet cells in the duodenum of calves subjected to milk volume restriction and supplemented with 2-hydroxy-4-(methylthio)butanoic acid (HMTBa). We used 21 Holstein mixed-breed calves, aged between 3 and 15 d with an average weight of 36.8 kg, and housed in pens with troughs for hay, concentrate, and water. The study included two consecutive experimental periods (first period [P1] and second period [P2]) of 21 d each, with 7 d of adaptation to the diet and facilities. The calves were distributed in a completely randomized design in three treatments with seven repetitions. 1) Control: 6 liters of milk/d during P1 and 6 liters of milk/day during P2; 2) RES (milk restriction): 3 liters of milk/day during P1 and 6 liters of milk/day during P2; and 3) RES + HMTBa: 3 liters of milk/day during P1 and 6 liters of milk/day during P2 + 3.3 g of HMTBa/day in both periods. HMTBa was supplied in milk, and the amount of concentrated ration and hay provided and leftovers were recorded daily to estimate dry matter (DM) and crude protein consumption. Mean daily weight gain (DWG), final weight (FW), and feed conversion (FC) were obtained at the beginning and at the end of each 21-d period. Plasma concentrations of ghrelin and leptin, triglycerides, total protein, urea, lactate, creatinine, alkaline phosphatase, and cholesterol were measured for P1 and P2 at the end of each 21-d period. At the end of P2, animals were slaughtered; sections of the duodenum were collected to evaluate the expression of GHS-R1a and quantity of goblet cells; hypothalamus was used to evaluate the expression of GHS-R1a; rumen was used to evaluate the thickness of epithelium and keratin and the density, height, and width of ruminal papillae. In P1, total DM consumption, FW, DWG, glucose, and triglycerides were lower in the RES and RES + HMTBa groups (P < 0.001). In P2, there was an improvement in the FC of the RES + HMTBa group (compared with Control and RES groups) and a lower urea concentration in the RES group (compared with Control and RES + HMTBa groups) (P < 0.001). No differences were observed among groups regarding hormonal concentrations, histological parameters, and GHS-R1a expression in the duodenum and hypothalamus. Therefore, milk restriction combined with HMTBa supplementation promoted greater compensatory gain by a mechanism independent of changes in GHS-R1a expression and hormone levels of ghrelin and leptin.     

前列腺素F_（2α）受体抑制剂和Ghrelin在奶牛繁殖中的应用前景

奶牛循环系统中高浓度前列腺素F_（2α）（PGF_（2α））是导致早期胚胎死亡的主要原因之一,而早期胚胎死亡又是影响奶牛繁殖力降低的主要因素。最近,牛胚胎中PGF_（2α）受体的发现和定位及PGF_（2α）受体抑制剂降低PGF_（2α）对胚胎发育抑制作用等。研究结果表明,PGF_（2α）受体抑制剂在奶牛繁殖中具有广阔的应用前景。Ghrelin作用在奶牛生殖过程的机理尚不完全清楚,但青年奶牛生殖器官、卵母细胞和早期胚胎中均能检测到Ghrelin、GHSR-1a mRNA及其表达蛋白等。一系列研究结果预示,Ghrelin在奶牛生殖过程中可能作为重要的代谢信号直接参加调控生殖激素的分泌、胚胎发育、生殖道营养物质的运输和生殖道功能的维持等生理过程。     

脑肠肽ghrelin的研究进展

介绍了近年来发现的由28个氨基酸残基组成的ghrelin的来源、分子结构、不同物种间的ghrelin组成差异、分布组织及存在形式，归纳了影响ghrelin分泌的因素，认为ghrelin在调节生长激素的释放、能量代谢、其他激素的分泌和参与调节睡觉一觉醒模式及细胞增殖方面发挥了重要生物学效应。