Leptin Molecular Regulatory Mechanisms That Prevent Growth hormone Deficiency

 描述（由申请人提供）：脂肪因子激素，瘦素告知靶细胞有关身体脂肪储存和代谢状态。瘦素信号传导的一个关键功能反应是垂体促生长素分泌的生长激素（GH）增加。瘦素信号传导的丧失导致GH缺乏症（GHD），伴随着代谢紊乱，包括成人发病肥胖、儿童骨生长丧失、低骨密度、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依据。介导瘦素信号传导至体细胞生长激素分泌的机制尚不清楚，这代表了一个主要的知识差距，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对生长激素功能的潜在机制，我们采用Cre-loxP技术选择性地在生长激素细胞中消除小鼠瘦素受体（Lepr外显子1的floxed等位基因）。这个模型使我们能够研究瘦素信号传导的作用，特别是在一个动物的生长激素，否则是健康的。我们已经描述了成年发病GHD和肥胖和随之而来的代谢功能障碍，在这些小鼠。我们最近引入了一个Cre-重组酶报告转基因到模型中，允许通过荧光鉴定生长激素的研究。该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通过减弱miRNA指导的GH mRNA翻译和GH蛋白积累的转录后控制来介导的。这一假设是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GH蛋白的积累，这表明GH分泌的缺陷是由于mRNA翻译控制的失调。我们已经确定了GH mRNA 3'非翻译区（3' UTR）内多个miRNA的结合位点，其中3个在突变小鼠的垂体中特异性升高。然而，我们还没有证明这些变化是专门在生长激素。因此，总体目标是确定候选miRNAs是否参与瘦素或饥饿素信号传导至促生长素。在Subaim 1中，我们将使用我们新开发的模型进行逐步实验，旨在确定突变体促生长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FACS。Subaim 2评估了选择性阿托莫西米在恢复GH储存中的疗效，还将包括一种无偏倚的方法来鉴定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miRNA来恢复突变体促生长素中的GH激素储存。本研究将探讨瘦素和生长激素释放肽在优化生长激素功能和预防生长激素缺乏症中的具体机制。Antagomir拯救实验可能会导致未来的GHD治疗方法。

 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.