Leptin Molecular Regulatory Mechanisms That Prevent Growth hormone Deficiency

预防生长激素缺乏的瘦素分子调节机制

• 批准号: 8968138
• 负责人: GWEN V CHILDS
• 金额: $ 7.45万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-10 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8968138
• 关键词: 3' Untranslated Regions; Address; Adult; Alleles; Animals; Attenuated; Binding Sites; Biological Assay; Body fat; Bone Density; Bone Growth; Cells; Child; Data; Event; Exons; Fluorescence; Frozen Sections; Functional disorder; Future; Genetic Translation; Homeostasis; Hormones; In Situ Hybridization; Knock-out; Knockout Mice; Knowledge; Lasers; Lead; Leptin; Link; LoxP-flanked allele; Mediating; Messenger RNA; Metabolic; Metabolic Diseases; MicroRNAs; Microscopy; Mission; Modeling; Molecular; Mus; Mutant Strains Mice; Obesity; Pilot Projects; Pituitary Gland; Play; Population; Post-Transcriptional Regulation; Prevention; Proteins; Public Health; Publishing; Regulation; Regulatory Element; Reporter; Reporter Genes; Research; Role; Signal Transduction; Somatotropin; Source; Technology; Testing; Therapeutic; Transgenes; Translational Repression; attenuation; base; cell type; comparative; design; disorder prevention; frailty; ghrelin; growth hormone deficiency; growth hormone-releasing hormone receptor; innovation; insight; leptin receptor; mouse model; muscle form; mutant; new therapeutic target; novel; novel diagnostics; novel therapeutics; prevent; public health relevance; recombinase; research study; response; restoration

 DESCRIPTION (provided by applicant): The adipokine hormone, leptin informs target cells regarding body fat stores and metabolic status. A key functional response to leptin signaling is an increase in growth hormone (GH)-secretion by pituitary somatotropes. Loss of leptin signaling results in GH deficiency (GHD), with attendant metabolic disorders including adult onset obesity, loss of bone growth in children, low bone density, loss of lean muscle mass and frailty in adults. An understanding of the mechanisms of leptin-induced GH secretion would provide a basis for prevention and correction of these metabolic disorders. The mechanisms mediating leptin signaling to somatotrope GH-secretion are unknown, representing a major knowledge gap that will be addressed in the pilot studies described in this application. To study the mechanisms underlying leptin signaling to somatotrope function, we employed Cre-loxP technology to ablate the murine leptin receptor (floxed alleles of Lepr exon 1) selectively in somatotropes. This model allows us to study the role of leptin signaling specifically in somatotropes in an animal that is otherwise healthy. We have described the adult onset GHD and obesity and consequent metabolic dysfunctions in these mice. We recently introduced a Cre-recombinase reporter transgene into the model, to allow studies of somatotropes identified by fluorescence. The objective of this pilot study is to utilize this newly validated cellular mode to test our central hypothesis that leptin signaling to control somatotrope function is mediated through the attenuation of miRNA- directed, post-transcriptional control of GH mRNA translation and GH protein accumulation. This hypothesis was based on our recently published study indicating that the mutant somatotropes have normal levels of GH mRNA, but reduced GH protein accumulation, suggesting that the deficit in GH secretion is due to de-regulation of mRNA translational control. We have identified binding sites for multiple miRNAs within the GH mRNA 3' untranslated region (3' UTR), 3 of which were specifically elevated in pituitaries from mutant mice. However, we have not proven that these changes are specifically in somatotropes. Thus, the overall aim is to determine if candidate miRNAs are involved in leptin or ghrelin signaling to somatotropes. In Subaim 1, we will use our newly developed model to perform step-wise experiments designed to determine if mutant somatotropes have higher levels of candidate miRNAs. Multiple approaches will validate the short-term culture and FACS. Subaim 2 assesses the efficacy of selective antagomirs in the restoration of GH stores and will also include an unbiased approach to identify the region within the Gh mRNA that mediates translational control. Subaim 3 will determine if Ghrelin restores GH hormone stores in mutant somatotropes by attenuating candidate miRNAs. This pilot study will explore specific mechanisms used by leptin and ghrelin in the optimization of somatotrope function and the prevention of GH deficiency. Antagomir rescue experiments may lead to future therapeutic approaches for GHD.

 描述（由申请人提供）：脂肪激素瘦素向靶细胞通报身体脂肪储存和代谢状态，对瘦素信号传导的关键功能反应是垂体生长激素导致生长激素（GH）分泌增加。生长激素缺乏症（GHD），伴有代谢紊乱，包括成人肥胖、儿童骨骼生长丧失、骨密度低、瘦肌肉质量丧失和成人虚弱。瘦素诱导的 GH 分泌机制将为预防和纠正这些代谢紊乱提供基础。介导瘦素信号传导至生长激素 GH 分泌的机制尚不清楚，这代表了一个主要的知识空白，将在本申请中描述的试点研究中解决。为了研究瘦素信号传导至生长激素功能的机制，我们采用 Cre-loxP 技术选择性地消除小鼠瘦素受体（Lepr 外显子 1 的 floxed 等位基因）。该模型使我们能够研究瘦素信号在健康动物的生长激素中的作用，我们已经描述了这些小鼠的成年 GHD 和肥胖以及随之而来的代谢功能障碍。进入模型，以便研究通过荧光识别的生长激素。这项初步研究的目的是利用这种新验证的细胞模式来测试我们的中心假设，即控制生长激素功能的瘦素信号传导是介导的。通过减弱 miRNA 指导的 GH mRNA 翻译和 GH 蛋白积累的转录后控制，这一假设基于我们最近发表的研究，表明突变体生长激素具有正常水平的 GH mRNA，但减少了 GH 蛋白积累。 GH 分泌缺陷是由于 mRNA 翻译控制失调所致，我们已经鉴定出 GH mRNA 3' 非翻译区 (3' UTR) 内多个 miRNA 的结合位点，其中 3 个在 GH mRNA 3' 非翻译区 (3' UTR) 中特异性升高。然而，我们尚未证明这些变化是特定于生长激素的，因此，总体目标是确定候选 miRNA 是否参与生长激素的瘦素或生长素释放肽信号传导。模型进行逐步实验，旨在确定突变生长激素是否具有更高水平的候选 miRNA。多种方法将验证短期培养和 FACS 评估选择性的功效。 Subaim 3 还将采用一种公正的方法来识别 Gh mRNA 中介导翻译控制的区域，以确定 Ghrelin 是否通过减弱候选 miRNA 来恢复突变型生长激素中的 GH 激素储存。瘦素和生长素释放肽在优化生长激素功能和预防 GH 缺乏方面使用的特定机制可能会导致未来的治疗方法。 GHD。