The Essential Role of Endogenous Ghrelin in Growth Hormone Expression during Zebrafish Adenohypophysis Development

Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development. (Endocrinology 150: 2767-2774, 2009)

Motilin/ghrelin荷尔蒙基因家族及其受体基因家族的分子进化研究和云南闭壳龟（Cuorayunnanensis）的分子鉴定及进化地位研究

哺乳动物motilin/ghrelin荷尔蒙基因家族编码产生三种多肽荷尔蒙，motilin、ghrelin和obestatin。这三种荷尔蒙分别结合各自特异性受体MLNR、GHSR和GPR39，调控不尽相同，但类似或相关的生理生化过程，并且它们的受体相互之间也高度同源。根据达尔文的进化论，任何复杂的生物系统都是在原有基础上，在自然选择的作用下逐步调整优化而来（Darwin 1859）。一方面，一个系统中相互作用的各个组分在进化过程中同时发生或变化的概率微乎其微；另一方面，紧密相互关联的系统中一个组分的孤立存在看起来又是毫无意义的。本研究中，我们基于系统发育分析重建了荷尔蒙基因家族及其受体基因家族的进化历史，探讨它们在进化中的关系。从而了解在一个整合的系统中，基因复制（gene duplication）后产生的新的组分是如何演化的，是如何形成新的分子间相互作用（如荷尔蒙–受体间特异性相互作用）和发生功能分化的。 我们的研究结果表明，preproghrelin（GHRL）和prepromotilin（MLN）源于一个祖先基因，是由基因重复而来，基因重复发生在C端两个新的翻译后剪辑位点演化出现之后，既两栖动物与羊膜动物分歧之后（而后产生了新的多肽荷尔蒙，例如高等哺乳动物GHRL中的obestatin）。受体与配体的进化历史并不一致。受体GPR39最先分歧，然后类似GHSR的祖先基因经历先后两次重复，产生了硬骨鱼世袭特异的基因簇A，MLNR和GHSR，基因重复事件的发生早于硬骨鱼与四足动物的分歧。Ghrelin/GHSR信号通路系统从硬骨鱼到哺乳动物的进化过程中十分保守，结构和功能几乎都没有发生太大变化。Motilin和MLNR间的特异性相互作用是荷尔蒙基因重复发生后，配体、受体间协同进化的结果，自此形成了新的Motilin/MLNR信号通路系统。 我们提出了一个复杂系统（分子间相互作用网络）进化的模型：基因重复或酶饰作用后产生了新的组分，它们通常是先前已有组分的一种结构上的变异，能与之前已经存在的分子形成新的分子间相互作用，从而演化出新的功能。基因重复之前，通常存在基因共享，即一种分子参与到多个过程、多种分子间相互作用。基因重复或酶饰作用生成了新组分，新的分子间相互作用，为功能上的专化和特化提供了条件。 此外我们还对新近发现的活的云南闭壳龟进行了分子鉴定，并探讨了云南闭壳龟的系统发育地位。云南闭壳龟（Cuora yunnanensis，Boulenger，1906）曾被认为已经灭绝，在保护生物学上受到广泛的关注。我们测定了三只活的云南闭壳龟线粒体COI和ND4及His、Ser、Leu tRNA序列片断，共1725碱基序列。结合闭壳龟属其它物种序列，包括之前测定的一只云南闭壳龟标本（MNHN 1907.10）的DNA序列，进行了分子系统学分析。与100年前的标本比较，无论是形态上、还是本文的分子系统结果都显示，新发现的活的云南闭壳龟确实是云南闭壳龟。同时，我们的结果确证了标本序列的可信性，揭示云南闭壳龟不是近期杂交形成的，代表了进化上独立的遗传谱系，且种内仍存在一定的遗传多样度。本文是分子系统学在濒危物种保护应用中的成功案例。我们的结果为推翻云南闭壳龟已经灭绝的观点提供了进一步强有力的分子生物学证据，但该物种极其稀少的状况提示其前景不容乐观，必须尽快采取有力的措施予以重点保护。

Laparoscopic greater curvature plication in morbidly obese women with type 2 diabetes:effects on glucose homeostasis, postprandial triglyceridemia and selected gut hormones

Background: Laparoscopic greater curvature plication (LGCP) is an emerging bariatric procedure that reduces the gastric volume without implantable devices or gastrectomy. The aim of this study was to explore changes in glucose homeostasis, postprandial triglyceridemia, and meal-stimulated secretion of selected gut hormones [glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), ghrelin, and obestatin] in patients with type 2 diabetes mellitus (T2DM) at 1 and 6 months after the procedure. Methods: Thirteen morbidly obese T2DM women (mean age, 53.2 ± 8.76 years; body mass index, 40.1 ± 4.59 kg/m2) were prospectively investigated before the LGCP and at 1- and 6-month follow-up. At these time points, all study patients underwent a standardized liquid mixed-meal test, and blood was sampled for assessment of plasma levels of glucose, insulin, C-peptide, triglycerides, GIP, GLP-1, ghrelin, and obestatin. Results: All patients had significant weight loss both at 1 and 6 months after the LGCP (p≤0.002), with mean percent excess weight loss (%EWL) reaching 29.7 ;plusmn2.9 % at the 6-month follow-up. Fasting hyperglycemia and hyperinsulinemia improved significantly at 6 months after the LGCP (p<0.05), with parallel improvement in insulin sensitivity and HbA1c levels (p<0.0001). Meal-induced glucose plasma levels were significantly lower at 6 months after the LGCP (p<0.0001), and postprandial triglyceridemia was also ameliorated at the 6-month follow-up (p<0.001). Postprandial GIP plasma levels were significantly increased both at 1 and 6 months after the LGCP (p<0.0001), whereas the overall meal-induced GLP-1 response was not significantly changed after the procedure (p ;gt0.05). Postprandial ghrelin plasma levels decreased at 1 and 6 months after the LGCP (p<0.0001) with no significant changes in circulating obestatin levels. Conclusion: During the initial 6-month postoperative period, LGCP induces significant weight loss and improves the metabolic profile of morbidly obese T2DM patients, while it also decreases circulating postprandial ghrelin levels and increases the meal-induced GIP response. © 2013 Springer Science+Business Media New York.