Leptin Molecular Regulatory Mechanisms That Prevent Growth hormone Deficiency

 描述(申请人提供)：脂肪因子激素，瘦素通知目标细胞有关身体脂肪储存和代谢状态。对瘦素信号的一个关键功能反应是垂体生长激素(GH)分泌增加。瘦素信号的缺失会导致生长激素缺乏症(GHD)，伴随而来的代谢紊乱包括成人发作性肥胖、儿童骨生长丧失、骨密度低、瘦肌肉质量丧失和成人虚弱。了解瘦素诱导GH分泌的机制将为预防和纠正这些代谢紊乱提供基础。瘦素信号转导生长激素分泌的机制尚不清楚，这是一个重大的知识缺口，将在本申请中描述的试点研究中解决。为了研究瘦素信号转导促生长激素功能的机制，我们利用Cre-loxP技术选择性地阻断了生长激素中的小鼠瘦素受体(Lepr外显子1的等位基因)。这个模型使我们能够研究瘦素信号的作用，特别是在生长激素中，在其他方面是健康的动物。我们已经描述了这些小鼠的成人发病、GHD和肥胖以及随后的代谢功能障碍。我们最近在模型中引入了Cre重组酶报告基因，以便于对荧光鉴定的生长激素进行研究。这项初步研究的目的是利用这一新验证的细胞模式来检验我们的中心假设，即瘦素信号控制生长激素功能是通过减弱miRNA指导的、转录后控制GH mRNA翻译和GH蛋白积累来介导的。这一假说是基于我们最近发表的一项研究，该研究表明突变的生长激素具有正常的GH mRNA水平，但减少了GH蛋白的积累，这表明GH分泌不足是由于mRNA翻译调控的去调节。我们已经在生长激素mRNA3‘非翻译区(3’UTR)中确定了多个miRNAs的结合位点，其中3个在突变小鼠的脑垂体中特异性上调。然而，我们还没有证明这些变化是生长激素所特有的。因此，总体目标是确定候选miRNAs是否参与瘦素或Ghrelin向生长激素传递信号。在Subaim 1中，我们将使用我们新开发的模型进行循序渐进的实验，旨在确定突变的生长激素是否具有更高水平的候选miRNAs。多种方法将验证短期培养和FAC。Subaim 2评估了选择性对映体在恢复生长激素储存方面的有效性，并将包括一种公正的方法来确定Gh mRNA中介导翻译控制的区域。Subaim 3将确定Ghrelin是否通过减弱候选miRNAs来恢复突变生长激素的生长激素储存。这项初步研究将探索瘦素和Ghrelin在优化生长激素功能和预防GH缺乏症中的具体作用机制。安塔科米尔的抢救实验可能会导致未来治疗GHD的方法。

 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.