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.     

绵羊生长激素(oGH)的分子克隆与序列测定

本文旨在新疆绵羊生长激素(oGH)基因的克隆。从阿勒泰×中国美利奴杂交羊脑垂体细胞中提取总RNA,分离得到具翻译活性的mRNA。用RTPCR方法扩增出编码oGH的基因,长度为671bp。将扩增产物经琼脂糖凝胶电泳后,回收纯化。采用PCR克隆试剂盒将扩增产物连接至pTAdv质粒上,经PstI和XbaI双酶切鉴定正反插入后,筛选正向插入阳性克隆,并进行序列分析。结果表明克隆到的oGH基因序列与国外报道的序列有4个碱基差异,但所推导的氨基酸序列完全一致。oGH基因的开放阅读框架共含654个核苷酸,编码217个氨基酸,其中信号肽26个氨基酸,编码碱基为1～78(78bp);成熟肽191个氨基酸,编码碱基为79～651(573bp);终止密码子TAG(652～654bp)。其蛋白质的氨基酸序列与牛、人和鱼的序列同源性分别为99.08%、26.7%和5.9%。     

Genetic diversity of growth hormone receptor gene in cattle

Growth hormone receptor (GHR) belongs to a member of the cytokine receptor superfamily. Polymorphism of presence or absence of an approximately 1.2 kbp LINE-1 element is observed in bovine GHR gene. The present study was carried out for estimating the genetic diversity and the origin of the LINE-1 element in 10 European, Southeastern Asian and East Asian cattle breeds or populations. Genotyping of the LINE-1 revealed predominant LINE-1 presence in European breeds (0.917∼0.991), absence in the Bos taurus indicus populations (0.000∼0.017), and intermediate presence in Northeast Asian cattle (0.417∼0.522). From genetic features of LINE families, LINE-1 of GHR could be attributed to the same origin in both European and Asian cattle, and Asian LINE-1 may not be derived from recent introgression. This result suggested that LINE-1 in bovine GHR gene could have arisen in an ancestral population of Bos taurus taurus .     

Effect of growth hormone on estrogen receptor binding properties in the chick oviduct in vivo

The equilibrium dissociation constant (K_d) and the maximum binding capacity (B_max) of estrogen receptor in cytosolic and nuclear fractions in oviduct of pullets following various daily injections (1–10 times) of growth hormone (GH: 50 µg/chick) were examined by Scathchard analysis of specific [³H]estradiol‐17β (E₂) binding. The K_d values of receptor in both fractions decreased after three times of GH‐injection. In the B_max values, three times of the injection caused a marked decrease in that value in the cytosolic fraction with a concomitant increase in that in the nuclear fraction, whereas the total B_max (sum of B_max in the cytosolic and nuclear fractions) did not change. A similar relationship between the K_d values in the binding of the two fractions was also observed in 4–10 times of GH‐injection. However, in 4–10 times of GH‐injection the B_max of the both fractions and total B_max was greater than that in vehicle‐injection (control). When the chicks were injected with 6–10 times of GH‐injection, the weight of oviduct was increased. No change in the plasma concentrations of E₂ was found following GH‐ and vehicle‐injections into the chicks. The results suggest that growth hormone stimulates the growth of the chick oviduct by increasing the binding affinity and capacity of estrogen receptor.     

Effects of fatty acid transport protein 1 on proliferation and differentiation of porcine intramuscular preadipocytes

Fatty acid transport protein 1 (FATP1) plays an important role in the fatty acid transmembrane transport and fat deposition. However, its role in porcine intramuscular preadipocytes proliferation and differentiation remain poorly understood. Here, we examined the effects of pFATP1 on porcine intramuscular preadipocytes proliferation and differentiation. Overexpression of pFATP1 in porcine intramuscular preadipocytes significantly promoted the proliferation of porcine intramuscular preadipocytes, and also significantly upregulated the expressions of peroxisome proliferator‐activated receptor γ, CCAAT enhancer binding protein α, lipoprotein lipase, fatty acid synthetase and perilipin 1. Moreover, overexpression of pFATP1 in porcine intramuscular preadipocytes significantly increased fat accumulation and downregulated β‐catenin protein expression. Overall, our results indicated that pFATP1 played an important role in porcine intramuscular preadipocytes proliferation and differentiation, and it might promote adipogenesis in porcine intramuscular preadipocytes by repressing Wnt/β‐catenin signaling pathway.     

Reduction in channel catfish hepatic growth hormone receptor expression in response to food deprivation and exogenous cortisol

The objective of this study was to assess the effects of food deprivation and exogenous cortisol administration on somatic growth of channel catfish, Ictalurus punctatus, and examine the resultant changes in circulating insulin-like growth factor-I (IGF-I) concentrations and growth hormone receptor (GHR) gene expression. Integral to this objective, we report the isolation, sequence, and characterization of channel catfish GHR. Sequence analysis and characterization results indicate sequence identity and tissue distribution similar to GHRs in other teleost fish and several functional characteristics conserved in known vertebrate GHRs. The effects of food deprivation and dietary exogenous cortisol administration were assessed as part of a 4-week study. Growth was significantly reduced after 4 weeks in cortisol-fed fish compared to fed-control fish, and fasting resulted in weight loss. At the end of the 4-week study, both IGF-I plasma concentrations and hepatic GHR mRNA abundance were significantly reduced in fasted and cortisol-fed catfish. Levels of hepatic GHR mRNA were positively correlated to circulating IGF-I levels. These results suggest that a reduction in hepatic GHR gene expression might serve as a mechanism for the reduction of circulating IGF-I and growth in channel catfish during periods of food deprivation and stress.     

Bos indicus type of growth hormone receptor gene is retained in Japanese Black cattle

The growth hormone receptor (GHR) gene is responsible for growth and carcass traits, and polymorphisms associated with the variation of meat production are thought to occur in the liver‐specific promoter of the GHR gene in cattle. The aim of this study was to analyse the structure of the liver‐specific promoter of GHR in Japanese Black cattle, as the relationship between GHR polymorphism and meat production is poorly understood in this breed. Typically in European cattle, the LINE‐1 element, a family of retrotransposons, is inserted in the liver‐specific promoter. However, a short GHR promoter without the LINE‐1 sequence was found in the Japanese Black breed as in Bos indicus cattle. The frequency of the short allele was approximately 60%. In addition, 24 of 29 Holstein/Japanese Black crosses carried the short allele from their sire. The present result suggests that the short allele for GHR may be a candidate marker for improving meat production of Japanese Black cattle.     

Predominant expression of growth hormone secretagogue receptor in the caudal lobe of chicken pituitary and parallel developmental increase in expression of pituitary GH and proventriculus ghrelin mRNA

To examine the involvement of ghrelin in growth hormone (GH) synthesis in the chicken pituitary, the regional distribution of GH secretagogue receptor (GHS‐R)/ghrelin receptor was investigated. Quantitative real‐time polymerase chain reaction (Q‐PCR) analysis revealed that the expression levels of GHS‐R and GH mRNA in the caudal lobe were about fourfold and sevenfold higher in the cephalic lobe of 7 day‐old chickens, respectively. Immunohistochemical analysis showed that GHS‐R immunoreactivity was more abundant in the caudal lobe than in the cephalic lobe, as was the case for GH immunoreactivity. By Q‐PCR, parallel increases were observed in the expression levels of ghrelin mRNA in the proventriculus and GH mRNA in the pituitary from embryonic day 17 to day 7 after hatching, whereas no significant change was found in the expression levels of GHS‐R mRNA in the pituitary during this period. These results suggest that proventriculus‐derived ghrelin may participate in pituitary GH synthesis by acting on its receptor during late embryonic development and the early post‐hatching period in chickens.     

Effect in sheep of dietary concentrate content on secretion of growth hormone, insulin and insulin-like growth factor-I after feeding

This study was designed to examine the effects of the proportion of concentrate in the diet on the secretion of growth hormone (GH), insulin and insulin‐like growth factor‐I (IGF‐I) secretion and the GH‐releasing hormone (GHRH)‐induced GH response in adult sheep fed once daily. Dietary treatments were roughage and concentrate at ratios of 100:0 (0% concentrate diet), 60:40 (40% concentrate diet), and 20:80 (80% concentrate diet) on a dry matter basis. Mean plasma concentrations of GH before daily feeding (10.00–14.00 hours) were 11.4 ± 0.4, 10.1 ± 0.5 and 7.5 ± 0.3 ng/mL on the 0, 40 and 80% concentrate diet treatments, respectively. A significant decrease in plasma GH concentration was observed after daily feeding of any of the dietary treatments and these decreased levels were maintained for 8 h (0%), 12 h (40%) and 12 h (80%), respectively (P < 0.05). Plasma IGF‐I concentrations were significantly decreased 8–12 h and 4–16 h after the end of feeding compared with the prefeeding level in the 40 and 80% concentrate diet treatments, respectively (P < 0.05). GHRH injection brought an abrupt increase in the plasma GH concentrations, reaching a peak 10 min after each injection, but, after the meal, the peak plasma GH values for animals fed 40% (P < 0.05) and 80% (P < 0.01) concentrate diet were lower than that for roughage fed animals. The concentrate content of a diet affects the anterior pituitary function of sheep resulting in reduced baseline concentrations of GH and prolonged GH reduction after feeding once daily.     

肌间脂肪沉积能力相关基因在前脂肪细胞分化前后的差异表达

研究表明,HBA、HBB、MB、HSPB1、MSRA、TCAP、TXN、PSMA1、GSTM1、COX6B1、VDAC1等基因在具有不同脂肪沉积程度的牛肉中存在差异表达,并对肌肉中脂肪的沉积情况具有潜在的影响。本研究以牛前体脂肪细胞为研究对象,诱导其分化为成熟脂肪细胞,通过实时荧光定量PCR检测上述基因在前体脂肪细胞分化前后的表达情况,通过油红O检测脂类沉积情况。结果表明,PSMA1、HBB、COX6B1、TCAP、MB、HBA、TXN、GSTM1、MSRA、HSPB1在前体脂肪细胞分化后表达量上升,而VDAC1表达量下降,前体脂肪细胞分化后脂质沉积明显加大,说明这些基因可能在调节前体脂肪细胞分化、脂质沉积中具有潜在作用。     

大鼠脂肪细胞分化过程中脂代谢相关基因转录表达时序的研究

对大鼠附睾和肾周脂肪组织分离的前体脂肪细胞原代培养，采用RT—PCR法分析脂代谢重要酶和转录因子基因在不同分化阶段转录表达的时序变化。结果显示，在前体脂肪细胞阶段，固醇调控元件结合蛋白（SREBP）-1c、碳水化合物反应元件结合蛋白（ChREBP）以及脂肪酸合成酶（FAS）、乙酰辅酶A羧化酶α（ACC1）、硬酯酰辅酶A去饱和酶（SCD）和激素敏感脂酶（HSL）基因均不转录表达；HSL mRNA在分化初期表达，中期表达水平最高，分化末期降低；SCD mRNA在分化中期表达，末期表达水平显著提高；分化初期FAS mRNA水平明显高于中期和末期；ACC1 mRNA仅在末期表达；分化初期SREBP-1c mRNA水平较高，中期和末期显著下降；ChREBP mRNA表达水平在分化末期稍高于中期，但差异不显著。表明，SREBP-1c mRNA的表达与脂肪细胞早期分化调控有关，分化成熟脂肪细胞的ChREBP mRNA表达可能与生脂基因的转录激活有关。     

Protein and mRNA expression of follicle‐stimulating hormone receptor and luteinizing hormone receptor during the oestrus in the yak (Bos grunniens)

Follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth, maturation and oestrus, but no clear pathway in the seasonal oestrus of yak (Bos grunniens) has been found. To better understand the role of FSH and LH in seasonal oestrus in the yak, six yaks were slaughtered while in oestrus, and the pineal gland, hypothalamus, pituitary gland, and gonads were collected. Using real‐time PCR and immunohistochemical assays, we determined the mRNA and protein expression of the FSH and LH receptors (FSHR and LHR) in these organs. The analysis showed that the FSHR mRNA expression level was higher in the pituitary gland tissue compared with LHR (p < .01) during oestrus. By contrast, there was low expression of FSHR and LHR mRNA in the pineal gland and hypothalamus. FSHR mRNA expression was higher than that of LHR (p < .05) in the ovary, whereas LHR mRNA expression was higher than that of FSHR (p < .01) in the uterus. FSHR and LHR proteins were located in the pinealocyte, synaptic ribbon and synaptic spherules of the pineal gland and that FSH and LH interact via nerve fibres. In the hypothalamus, FSHR and LHR proteins were located in the magnocellular neurons and parvocellular neurons. FSHR and LHR proteins were localized in acidophilic cells and basophilic cells in the pituitary gland, and in surface epithelium, stromal cell and gland epithelium in the uterus. In the ovary, FSHR and LHR protein were present in the ovarian follicle. Thus, we concluded that FSHR and LHR are located in the pineal gland, hypothalamus, pituitary and gonad during oestrus in the yak. However, FSHR was mainly expressed in the pituitary gland and ovaries, whereas LHR was mainly expressed in the pituitary gland and uterus.     

The effects of concentrations of lysine in media on differentiation of 3T3‐L1 preadipocytes

Intramuscular fat content is increased by feeding of low lysine diets in pigs. Reduction in dietary lysine intake results in low plasma lysine concentration and low cytosolic lysine concentration in skeletal muscles. From these observations, we hypothesized that low plasma lysine concentration in pigs fed on low lysine diets reduced supply of lysine from blood circulation to preadipocytes, and this limited supply of lysine might promote adipocyte differentiation in porcine muscles. In order to verify the hypothesis, we investigated the effects of low concentrations of lysine in culture medium on differentiation of 3T3‐L1 preadipocytes. Low concentration of lysine suppressed lipid accumulation and messenger RNA (mRNA) expression and enzyme activity of fatty acid synthase. mRNA expressions of peroxisome proliferator‐activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were lower in cells cultured in low lysine medium. On the other hand, mRNA and protein expressions of C/EBPβ and C/EBPδ were not inhibited by low concentrations of lysine in culture medium. These results indicate that low lysine concentrations in culture medium inhibit differentiation of 3T3‐L1 preadipocytes through inhibiting the mRNA expressions of PPARγ and C/EBPα.     

LH受体在山羊结状神经节中的分布

为了检测山羊迷走神经结状神经节中是否有促黄体生成素受体(LHR)的存在,探讨促黄体生成素(LH)是否能够影响结状神经节内脏感觉神经元的功能活动。本试验采用免疫组织化学SP法观察LHR在结状神经节中的分布特征,利用IPP 6.0图像半定量分析系统分析LHR在结状神经节中阳性神经元和阳性非神经元上的表达量差异。结果显示,LHR主要分布在神经元细胞膜和细胞质中,呈棕黄色为强阳性。在带核的神经元中,神经元细胞核呈圆形空泡状,LHR为阴性反应。神经元群间的神经纤维呈淡黄色,LHR为弱阳性。LHR在神经元胞体上的相对表达量极显著高于节内其他阳性非神经元结构(P0.01)。结果表明,山羊结状神经节内脏感觉神经元是LH作用的主要靶细胞,提示LH有可能通过作用于结状神经节内脏感觉神经元胞体上的LHR参与调节内脏器官感觉信息的传导。     

Toll-like receptor signaling is changed in ovine lymph node during early pregnancy

Toll-like receptors (TLRs) participate in regulation of adaptive immune responses, and lymph nodes play key roles in the initiation of immune responses. There is a tolerance to the allogenic fetus during pregnancy, but it is unclear that expression of TLR signaling is in ovine lymph node during early pregnancy. In this study, lymph nodes were sampled from day 16 of nonpregnant ewes and days 13, 16, and 25 of pregnant ewes, and the expressions of TLR family (TLR2, TLR3, TLR4, TLR5 and TLR9), adaptor proteins, including myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), and interleukin-1-receptor-associated kinase 1 (IRAK1), were analyzed through real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry analysis. The results showed that mRNA and protein levels of TLR2, TLR3, TLR4, TRAF6, and MyD88 were upregulated in the maternal lymph node, but TLR5, TLR9, and IRAK1 were downregulated during early pregnancy. In addition, MyD88 protein was located in the subcapsular sinus and lymph sinuses. Therefore, it is suggested that early pregnancy induces changes in TLR signaling in maternal lymph node, which may be involved in regulation of maternal immune responses in sheep.     

Effect of chromium nanocomposite supplementation on growth hormone pulsatile secretion and mRNA expression in finishing pigs

The study was conducted to investigate the effect of chromium nanocomposite (CrNano) on growth hormone (GH) pulsatile secretion and pituitary GH mRNA expression in finishing pigs. Fifty‐four crossbred pigs (65.57 ± 1.05 kg initial weight) were randomly allotted to one of the three treatments, with three replicate pens per treatment and six pigs per pen. Pigs were offered one of the four diets including a control diet or a control diet supplemented with 200 μg/kg chromium from either chromium chloride (CrCl₃) or CrNano for 35 days. During the trial, all pigs were given free access to feed and water. After completion of the feeding trial, blood samples were taken via auriculares at 15 min intervals for 3 h and three pigs from each treatment were slaughtered to collect pituitary to determine GH mRNA level. The results of GH dynamic secretion showed that supplemental CrNano increased the mean level, the lowest value, peak value and peak duration of GH significantly, while there was no significant effect on peak amplitude. Pituitary mRNA expression of GH was improved significantly in pigs fed diet containing Cr from CrNano. These results indicated that CrNano increased GH mRNA expression and secretion in finishing pigs.     

三价铬对肥育猪生长激素分泌及垂体mRNA表达的影响

将96头体质量约65 kg的杜长大三元杂交猪,按体质量相近、公母各半的原则随机分成4组,每组3个重复.试验猪1组设为对照,饲喂基础饲粮,其余3组为试验组,分别饲喂在基础饲粮基础上添加来源于氯化铬、吡啶羧酸铬,纳米铬的含200 μg/kg铬试验饲粮.试验猪充分饲喂,自由饮水,试验为期40 d.饲养试验结束前1 d,从每组中选择6头猪,间隔15 min颈静脉采血1次,连续3 h,制备血清用于生长激素动态分泌模式研究.饲养试验结束后,按体质量相近原则从每组中选择8头猪进行屠宰,测定背膘厚和背最长肌面积;并各采取3头猪的脑垂体,RT-PCR法测定生长激素mRNA水平.结果表明,饲粮中添加200μg/kg三价纳米铬使肥育猪日增重提高了6.31%(P<0.05),料重比降低了4.61%(P<0.05),背膘厚降低了24.32%(P<0.05),背最长肌面积提高了20.22%(P<0.05).生长激素动态模式分析结果显示,纳米铬组试验猪生长激素总体水平、最低值、峰值和峰持续时间分别提高了42.62%(P<0.05)、87.94%(P<0.05)、26.60%(P<0.05)和17.19%(P<0.05),吡啶羧酸铬组试验猪生长激素总体水平、峰值分别提高了36.58%(P<0.05)和27.18%(P<0.05).垂体生长激素基因RT-PCR分析结果显示,纳米铬组试验猪垂体生长激素mRNA水平提高了27.63%(P<0.05).研究结果提示,三价纳米铬可提高肥育猪垂体生长激素mRNA水平,促进机体生长激素分泌,从而促进生长和改善胴体特性.     

Growth hormone receptor (GHR) RNAi decreases proliferation and enhances apoptosis in CMT-U27 canine mammary carcinoma cell line

Canine mammary gland has been identified as a major site of the extrapituitary growth hormone (GH) production. This finding is linked to its role in tumourigenesis of the mammary gland. Our previous studies indicated the role of GH and GH receptor (GHR) in regulation of proliferation and apoptosis. Thus, we have optimized the ghr RNA interference method in canine mammary carcinoma cell line CMT-U27. We have analysed the effect of GHR reduction on the intracellular signalling and the cell cycle and apoptosis. The results showed that GHR reduction decreased the p-ERK1/2 expression and caused increase of apoptosis and decrease in number of cells at S and G2M phases. This study indicates that GHR besides proliferative effect promotes growth by increasing cell survival. It can tilt the balance between proliferation and death in cancer cells.     

Effects of different roughage sources and feeding levels on adipogenesis of ovine adipocytes

The objective of the present study was to conduct an adipogenic evaluation of different roughage sources and feeding levels during ruminant adipocyte differentiation in vitro. Six wether sheep were divided into a timothy hay feeding group (TFG, n = 3) and an Italian ryegrass straw feeding group (IFG, n = 3). The sheep were fed high‐roughage (HR), medium roughage (MR) and low‐roughage (LR) diets in a one‐way layout design each over a 6‐day period. Sheep serum samples collected on the last day of each dietary treatment were added to an adipogenic induction medium for differentiation of preadipocytes derived from sheep subcutaneous adipose tissue. The cytoplasmic lipid accumulations in the TFG serum‐treated preadipocytes were significantly higher than those of the IFG‐serum treated preadipocytes on day 12. Messenger RNA expression of CCAAT/enhancer‐binding protein (C/EBP)‐α, C/EBP‐β, C/EBP‐δ, fatty‐acid‐binding protein (aP2) and stearoyl‐coenzyme A desaturase (SCD) were regulated by each serum treatment. This study shows that different roughage source diets and roughage‐to‐concentrate ratio diets can regulate adipocyte differentiation via ruminant blood composition.