Endocrine Regulation of Fetal Adipose Tissue Metabolism in the Pig: Role of Hydrocortisone

Glucocorticoids have been shown to be essential for the excessive fat deposition and development of obesity in several animal models. This study was performed to characterize the role of glucocorticoids in the developmental regulation of adipose tissue metabolism. On day 70 of gestation, pig fetuses were hypophysectomized by micro-cauterization. Hypophysectomized fetuses were implanted subcutaneously with hydrocortisone pellets or received no hormone replacement. Fetuses were removed by laparotomy on day 90 of gestation. Additional fetuses were hypophysectomized on day 70, implanted with hydrocortisone pellets on day 90 and removed on day 105 of gestation. Several intact fetuses were also implanted subcutaneously with hydrocortisone pellets during this later gestational period. Serum cortisol concentrations were reduced in hypophysectomized pigs at both fetal ages and were restored to intact levels by hydrocortisone treatment. Hydrocortisone supplementation enhanced lipolytic response to isoproterenol in intact fetuses but failed to restore lipolytic response to isoproterenol in hypophysectomized animals at either fetal age. Hydrocortisone induced a slight increase in lipogenesis in hypophysectomized fetuses when administered from 70 to 90 days of gestation and a more dramatic increase when administered from days 90 to 105 of gestation. However, hydrocortisone had no effect on basal or insulin stimulated lipogenesis in intact fetuses when administered from days 90 to 105 of gestation. These results indicate that hydrocortisone may have a primary influence on adipose tissue metabolism during late fetal development only in the absence of inhibition from counterregulatory hormones of pituitary origin.     

豚鼠生长激素受体及其突变体在哺乳动物细胞中的功能性表达

采用RT PCR克隆得到豚鼠生长激素受体cDNA。序列同源比较显示生长激素受体的一些功能性保守氨基酸在豚鼠生长激素受体中被其他氨基酸所取代。例如 ,哺乳动物生长激素受体中保守的第 170位组氨酸和第 333位酪氨酸分别是受体二聚化和生长激素刺激蛋白质合成及脂生成所必需的 ,但在豚鼠生长激素受体中分别被酪氨酸和丝氨酸所取代。为此 ,采用定点突变法得到了突变体gpGHRY16 8H和 gpGHRS332Y ,并构建了表达质粒 pcDNA3 gpGHR ,pcDNA3 gpGHRY16 8H和pcDNA3 gpGHRS332Y。借助COS 7和CHO细胞 ,研究了豚鼠生长激素受体及其突变体的生物活性。实验表明转染了pcDNA3 gpGHR的COS 7细胞对牛生长激素具有高亲和性 [Ka=1.3× 10 9(mol/L) -1],并且用鼠抗生长激素受体单克隆抗体mAb2 6 3可检测到一分子量约 92kD的蛋白质。在CHO细胞中 ,虽然两个氨基酸的定点突变不影响受体与配体的结合 ,但都提高了生长激素刺激的蛋白质合成而降低生长激素刺激的脂肪生成。蛋白质印迹实验揭示突变体 gpGHRY16 8和gpGHRS332Y分别降低和提高了生长激素诱导的JAK2酪氨酸磷酸化。因此 ,报道了豚鼠生长激素受体在生长激素代谢功能中的调节作用以及受体中保守氨基酸的取代导致配体结合后信号转导的变化。     

Effects of Growth Hormone on the Function of β‐Adrenoceptor Subtypes in Rat Adipocytes

Objective: The influence of growth hormone (GH) on the regulation of lipolytic response to specific agonists to β‐adrenoceptors and several post‐receptor steps in the lipolytic cascade were investigated. Research Methods and Procedures: Adipose tissues from rats were incubated with or without GH (1.38 nM). After a 24‐hour incubation, isolated adipocytes were prepared for different assays. Rats were hypophysectomized. One week after operation, l‐thyroxine and hydrocortisone acetate was given to hypophysectomized rats. One group of rats was treated with GH (1.33 mg/kg, daily). After 1 week of hormonal treatment, adipose tissues were removed for different studies. Results: GH treatment increased both basal lipolysis and lipolytic sensitivity to dobutamine and CGP 12177 in adipocytes. The lipolytic sensitivity to terbutaline was not influenced by GH treatment. GH treatment increased the maximal lipolytic response to dobutamine and CGP 12177, but not to terbutaline as determined with absolute values of lipolysis. Forskolin‐induced lipolysis was increased by addition of GH to tissues. Moreover, GH treatment resulted in enhanced expression of hormone‐sensitive lipase. GH treatment in hypophysectomized rats influenced neither the expressions of Gαs protein and cholera toxin‐catalyzed adenosine diphosphate‐ribosylation of Gαs protein, nor cholera toxin‐induced 3′, 5′‐cyclic adenosine monophosphate accumulation. However, the expression of Gαi protein was decreased after GH treatment. Discussion: These and previous results suggest that GH increases lipolysis in rat adipocytes partly through the β‐adrenergic system, including increases in both β₁‐ and β₃‐adrenergic receptor function, and partly through enhanced adenylate cyclase function, and expression of hormone‐sensitive lipase, perhaps via a decrease in Gαi protein expression.     

猪生长激素cDNA在芽孢杆菌中的表达

利用随机克隆的枯草杆菌启动子-信号序列构建茅孢杆菌分泌载体pUS186。用限制酶将切除了信号序列的猪生长激素cDNA从质粒pLY3-PGH 604切下,亚克隆至pUS186,并在该cDNA的下游接上地衣杆菌α-淀粉酶基因的转录终止子,构建猪生长激素表达质粒pSGH 1864,将此质粒转化蛋白酶双缺陷的枯草杆菌DB104及短小茅孢杆菌289。SDS-聚丙烯酰胺凝胶电泳检出在发酵上清液中多出一条22kD的蛋白带,抗猪生长激素血清免疫印迹法证明这一蛋白带具有免疫活性,表明猪生长激素cDNA已在枯草杆菌及短小茅抱杆菌中表达。     

猪生长激素基因表达质粒的构建及其转基因动物研究

将猪生长激素基因（ＰＧＨ）克隆到质粒ｐＵＣ１９上,经酶切分析,确定其酶切图谱．把ＰＧＨ转录起始位点以前的序列切掉,换上羊ＭＴ－１ａ基因的启动子,构建成可以调控的表达载体ｐＳＭＴＰＧＨ,用于转基因动物的研究．采用微注射法将线状ｐＳＭＴＰＧＨ导入猪、鼠和金鱼的受精卵中,得到了相应的转基因动物．对这些动物鉴定分析表明,外源基因整合率因动物不同而异,但该基因在这３种动物中的整合率均在９％以上,其生长速度都高于对照组．     

Body Composition and Tissue Distributions in Growth Hormone Deficient Adults Before and After Growth Hormone Treatment

This study examines short and long-term effects of recombinant human growth hormone (rhGH) on body composition and regional tissue distributions by using a multicompartment technique based on computed tomography. Part I includes nine subjects aged 46 ± 9 years with adult onset GH deficiency who were examined before and in the end of 6 months treatment with rhGH (0.4 U. kg^?1. week^?1) in a double-blind crossover trial. Part II is an ongoing open trial including seven of the males in part I. They were treated with rhGH (0.25 U. kg^?1. week^?1) over an additional period of 24 months. Adipose tissue (AT) was reduced by 4.7 kg (p<0.01) while the muscle plus skin compartment (M) and visceral organs (V) were increased by 2.4 (p<0.05) and 0.7 kg (p<0.01), respectively, over 6 months of treatment with a high rhGH dose. A preferential lipid mobilization occurred in the visceral and subcutaneous trunk depots resulting in a changed AT distribution. Muscles of legs and arms increased while the increase of trunk muscles did not reach significance. The body composition changes were maintained over 2 years additional treatment. The preferential loss in visceral AT was further pronounced while other changes in tissue distributions observed during the first 6 months tended to be reversed on the lower rhGH dosage. It is concluded that growth hormone has profound and discordant effects on AT, M and V and with associated changes in tissue distributions. The beneficial effects on body composition seen in short-term treatment is preserved throughout an additional 24 months period of treatment.     

豚鼠生长激素受体cDNA的克隆

报道了豚鼠肝生长激素受体（ＧＨＲ）的ｃＲＮＡ克隆和编码区序列。它由１８９９ｂｐ组成,编码６１０个氨基酸。此外,还报道豚鼠ＧＨＲ的结构特征和同源性比较的结果。     

两栖和爬行类生长及生长激素分泌活动的调节

根据生长内分泌学,综述了近年来两栖和爬行类生长激素（ＧＨ）分泌活动的调节及生长激素在两栖爬行类生长中作用这一研究领域所取得的主要成就和研究进展,研究结果表明：在脊椎动物的进化过程中,ＧＨ的化学结构和功能是相当保守的;ＧＨ对两栖爬行类的生长起促进作用;类胰岛素生长因子（ＩＧＦｓ）也能冰分传递ＧＨ促进两栖类的生长;在两栖爬行类,下丘脑对ＧＨ分泌的调控较哺乳类缺乏特异性,利用外源ＧＨ促进两栖爬行类的生长     

猪生长激素基因在杆状病毒载体系统中的表达

通过对猪生长激素（ｐＧＨ）基因的ｃＤＮＡ进行测序,得到ｐＧＨｃＤＮＡ的全序列,并与Ｓｅｅｂｕｒｇ等报道的序列进行了比较和讨论。然后利用具人工合成启动子和多角体蛋白ＸＩＶ启动子的转移载体质粒ｐＳＸＩＶＶＩ＾＋Ｘ３／４构建出含ｐＧＨ基因的重组质粒ｐＸ３／４－ｐＧＨ。将ｐＸ３／４－ｐＧＨ与致死缺失型线性化ＡｃＭＮＰＶ－ＯＣＣ＾－ＤＮＡ共转染Ｓｆ９细胞,构建出既能形成多角体又能表达ｐＧＨ基因的苜蓿丫纹夜蛾     

黄绿木霉对温室番茄生长发育和内源激素的调控效应(英文)

利用黄绿木霉T1010制剂(Trichoderma aureviride 1010)处理多年连作番茄的日光温室土壤,探索其对日光温室番茄生长发育的影响及其机理.结果显示:与常规生产区相比,黄绿木霉T1010处理的日光温室番茄根、茎、叶的生长和果实发育指标都得到不同程度的改善,其主根长、侧根长、侧根数和单株根系干重分别提高13.15%、28.21%、49.40%、2.89%,单株茎干重、功能叶长、单功能叶干重和单株叶干重分别提高114.93%、14.17%、42.69%、29.94%,单株果穗数、已采收果穗数、单果穗座果数、单果重和小区产量分别提高9.33%、389.19%、37.60%、30.46%、20.00%,而第1果穗高度则降低19.45%,且除单株根系干重和单株果穗数外均达到极显著水平(P<0.01);相应番茄植株功能叶内源激素水平均极显著同步提高,低温胁迫下的赤霉素(GA)、玉米素核苷(ZR)、吲哚乙酸(IAA)和脱落酸(ABA)分别提高42.10%、33.16%、60.97%、119.40%.研究发现,黄绿木霉T1010可能主要是通过调控日光温室番茄的内源激素水平来有效改善其根、茎、叶的生长和果实发育状况,促进秋冬季日光温室番茄的可持续生产.     

Effect of Carbohydrate Overfeeding on Whole Body and Adipose Tissue Metabolism in Humans

Objective: To evaluate the effect of a 4‐day carbohydrate overfeeding on whole body net de novo lipogenesis and on markers of de novo lipogenesis in subcutaneous adipose tissue of healthy lean humans. Research Methods and Procedures: Nine healthy lean volunteers (five men and four women) were studied after 4 days of either isocaloric feeding or carbohydrate overfeeding. On each occasion, they underwent a metabolic study during which their energy expenditure and net substrate oxidation rates (indirect calorimetry), and the fractional activity of the pentose‐phosphate pathway in subcutaneous adipose tissue (subcutaneous microdialysis with 1, 6¹³C₂, 6, 6²H₂ glucose) were assessed before and after administration of glucose. Adipose tissue biopsies were obtained at the end of the experiments to monitor mRNAs of key lipogenic enzymes. Results: Carbohydrate overfeeding increased basal and postglucose energy expenditure and net carbohydrate oxidation. Whole body net de novo lipogenesis after glucose loading was markedly increased at the expense of glycogen synthesis. Carbohydrate overfeeding also increased mRNA levels for the key lipogenic enzymes sterol regulatory element‐binding protein‐1c, acetyl‐CoA carboxylase, and fatty acid synthase. The fractional activity of adipose tissue pentose‐phosphate pathway was 17% to 22% and was not altered by carbohydrate overfeeding. Discussion: Carbohydrate overfeeding markedly increased net de novo lipogenesis at the expense of glycogen synthesis. An increase in mRNAs coding for key lipogenic enzymes suggests that de novo lipogenesis occurred, at least in part, in adipose tissue. The pentose‐phosphate pathway is active in adipose tissue of healthy humans, consistent with an active role of this tissue in de novo lipogenesis.     

猪生长激素及其单克隆抗体的制备与纯化

本文介绍一种简便快捷提纯猪生长激素的方法。通过杂交瘤技术,首次获得抗猪生长激素的单克隆抗体,并讨论和比较从腹水纯化单克隆抗体的各种方法。     

ATGL调节细胞内脂质代谢和代谢相关疾病

脂肪组织甘油三酯水解酶(adipose triglyceride lipase, ATGL)是一种催化甘油三酯第一步水解的重要脂肪酶,在机体能量代谢调节中发挥重要作用.本文介绍了ATGL的基因和蛋白质结构,并详细综述了ATGL的功能调控和与其相关联疾病的研究进展,最后通过与激素敏感脂肪酶(HSL)比较,对ATGL的特征进行总结.     

氯化镧对牛血清谷胱甘肽过氧化物酶、生长激素、胰岛素、三碘甲腺原氨酸和四碘甲状腺原氨酸浓度的影响

选用36头平均体重420 kg,年龄2.5岁的中国西门答尔阉牛,采用随机区组设计分为4组,以混合精料和风干玉米秸秆为基础日粮,研究日粮中添加不同水平氯化镧对西门答尔阉牛营养物质消化、血清谷胱甘肽过氧化物酶、生长激素、胰岛素、三碘甲腺原氨酸和四碘甲状腺原氨酸浓度的影响。结果表明:实验至第60天时,每天添加0.9 g氯化镧的饲粮有机物质、粗蛋白、粗脂肪、粗纤维、中性洗涤纤维和酸性洗涤纤维的表观消化率较对照组和其它处理组分别提高了8.09%、7.13%、15.20%、9.47%、13.09%和20.48%,血清谷胱甘肽过氧化物酶活性较对照组和其它处理组显著提高了8.22、3.95和5.04个单位,血清生长激素、胰岛素浓度、三碘甲腺原氨酸和四碘甲状腺原氨酸浓度均显著高。     

外源猪生长激素基因在金鱼体内的整合与表达研究

自1982年Palmiter^[1]等人给小鼠受精卵雄原核注射大鼠生长激素基因培养成功“超级”鼠以来,由于人们认识到转基因技术导致动植物品种定向改良以及利用转基因动物作为生物反应器,大量合成和分泌贵重而安全的基因工程产品,因此转基因动物技术得到了迅速发展,相继开展了兔、鱼、猪、鸡、牛、羊等动物的转基因研究,并取得了一定的结果^[2,3,4]。但是在上述具有经济价值的高等脊椎动物中从事转基因研究,成本高、操作困难,而金鱼属于低等脊椎动物．具有产卵多、易获得同步卵、可控制体外受精和体外发育等特点,因而成为转基因动物研究的方便材料。生长激素是动物脑下垂体前叶分泌的单链多肽⑸,生理功能是促进碳水化合物代谢及核酸、蛋白质合成,整体效应表现为动物生长。本文以金鱼为实验材料,探讨猪生长激素基因导入其受精卵后整合、表达以及生物效应等问题,为进一步研究外源基因在高等动物内整合和表达调控机理提供参考。     

油菜生长发育期间内源激素含量的变化

初花期至盛花期华双3号、华杂4号、恢5900等3个品种花蕾的iPAs含量呈明显上升趋势,而秦油2号则刚好相反(表2)。对双低品种华双3号功能叶的研究表明：苗期至盛花期IAA含量始终最高,其后依次是GA1 3;、iPAs、ABA。功能叶IAA的含量在越冬期处于整个生育期的最低水平,蕾薹期达到高峰,随后下降：GA1 3和iPAs含量随生育进程逐渐升高,并在初花期达到峰值;ABA在苗期及抽薹后一直处于很低水平,但在越冬期达到最高值(图1、2)。蕾薹期IAA主要分布在幼嫩和正在生长的器官中并起重要作用,而在功能叶中相对要少;幼叶GA1 3和iPAs含量最高,功能叶其次,花蕾最低(表1)。角果皮与籽粒的内源激素含量差异极显著,在开花后第39天,籽粒中IAA、GA1 3;、iPAs的含量大大超过角果皮(表3)。结果 表明iPAs在促进籽粒的充实饱满、物质积累转化方面发挥重要作用。     

Ontogeny of the Response to Thyroxine (T4) in the Porcine Fetus: Interrelationships Between Serum T4, Serum Insulin-Like Growth Factor-1 (IGF-1) and Differentiation of Skin and Several Adipose Tissues

The temporal aspect of thyroxine (T₄) enhanced development of several fetal adipose tissue depots (locations) and skin was examined in the present study. On day 70 of gestation pig fetuses were either hypophysectomized (hypox), hypoxed and implanted with T₄ pellets or left intact (control). Subsequently, fetuses were removed for study at either 73, 75, 80, 85 and 90 days of gestation. Serum levels of T₄ and insulin-like growth factor I (IGF-I) were significantly elevated by T₄ after 3 days of treatment and throughout the study. T₄ treatment enhanced development of skin, inner and outer subcutaneous (SQ) adipose tissue and adipocyte lobules around hair follicles (HFL's) but degree of response and response time were tissue or depot dependent. The T₄ response time was shortest for skin and HFL's, longest for inner SQ and intermediate for the outer SQ. Considering the entire treatment period, magnitude of the T₄ response was much greater for skin and HFL's than for outer and inner SQ tissues. Possibly, these developmental characteristics indicate that T₄ directly influences skin and HFL's and indirectly influences inner SQ adipose tissue by increasing production of a locally produced adipogenic growth factor. Furthermore, developmental characteristics (morphological) indicated that putative local growth factor mediation of T₄ action may depend on stage of development or maturity of the tissue. IGF-I is clearly the most logical candidate as a putative local mediator of T₄, but evidence at the molecular level is needed to prove this suggestion. Regardless, the particular adipose tissue location or depot is an important aspect of hormone responsiveness during the critical or sensitive period of fetal adipose tissue development.     

微球包裹pcDNA3-GRF（1-32）在小鼠肌肉组织的表达及对生长的影响

以乳酸-乙醇酸共聚物为载体,采用复乳液中蒸发法制备载生长激素释放因子（Growth hormone releasing factor,GRF）真核表达质粒pcDNA3-GRF（1-32）微球,其中包封率达69%,平均粒径为2.20μm,载药量80μg/mg,收率70%;体内转染小鼠肌肉组织, 提取注射部位肌肉组织DNA 和总RNA,经PCR和RT-PCR,发现注射pcDNA3-GRF（1-32）微球组的GRF表达水平最高（UVIBAND Version 99分析）,释放的GRF表达质粒在小鼠肌肉内存在并表达的时间至少30d。体重统计结果表明,30d后微球包裹质粒组累积增重与其它组相比差异极显著（P<0.01）,比裸质粒组、质粒和空白微球混合物组、生理盐水组分别高12.87%,19.72%,58.58%;结论认为,载pcDNA3-GRF（1-32）微球具有缓释作用,并可实现GRF基因体内局部基因转染、表达,发挥其相应的生物学效应,有希望成为提高质粒在动物肌肉组织表达效率的新方法。     

The Adipose Tissue Phenotype of Hormone‐Sensitive Lipase Deficiency in Mice

Objective: To directly ascertain the physiological roles in adipocytes of hormone‐sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. Research Methods and Procedures: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. Results: Homozygous HSL^?/? mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild‐type and deficient littermates. HSL‐deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL^?/? mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL^?/? adipocytes, lipolysis is not significantly increased by β₃‐adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL^?/? adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48‐hour period at 4 °C was similar in HSL^?/? mice and controls. Overnight fasting was well‐tolerated clinically by HSL^?/? mice, but after fasting, liver triglyceride content was significantly lower in HSL^?/? mice compared with wild‐type controls. Conclusions: In isolated fat cells, the lipolytic rate after β‐adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL^?/? adipocytes suggests that HSL‐independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.