Role of the hormone LEAP2 in eating, body weight, blood glucose and survival

项目摘要 激素酰基-胃饥饿素作为进食、体重、血糖和存活的关键调节剂， 与其受体GHSR结合在过去的项目期间，我们研究了ghrelin分泌的调节。一个主要 研究发现，在模拟饥饿的严格热量限制方案下，ghrelin细胞表达的GSH 1， 肾上腺素能受体（ARs）介导生长素释放肽的分泌，而生长素释放肽又能防御明显的低血糖 and mortality.此外，在最近鉴定肝脏富集的抗微生物肽2（LEAP 2）作为一种抗微生物肽之后， 内源性GHSR拮抗剂，我们扩展了我们的研究，在细胞水平上表征LEAP 2功能 并测定人受试者和小鼠中由于各种代谢扰动引起的血浆LEAP 2变化。 我们发现LEAP 2既能使酰基-生长素释放肽超极化，又能阻止酰基-生长素释放肽激活弓状神经肽Y神经元， 这表明LEAP 2既作为GHSR反向激动剂又作为拮抗剂。我们还发现血浆 LEAP 2受代谢状态调节：其水平随肥胖和血糖升高而升高，随血糖升高而降低。 禁食和减肥这些变化大多与酰基-ghrelin相反。总的来说，这导致 我们提出了LEAP 2功能的以下模型：1）在肥胖状态下，LEAP 2升高而酰基-生长激素释放肽福尔斯下降， 使血浆LEAP 2/酰基-胃饥饿素摩尔比升高，从而限制酰基-胃饥饿素恶化的能力， 通过增加食物摄入、体重和血糖来治疗肥胖和葡萄糖耐受不良。2)物质以营养 缺乏状态，如严重的热量限制引起的，LEAP 2的下降创造了一个环境， 其中升高的酰基-胃饥饿素可以最有效地防止危及生命的低血糖症。在当前 R 01建议，我们测试这个模型在3个目标，通过使用大多是新颖的工具组合删除，击倒， 和/或在肥胖和严重热量限制的情况下中和LEAP 2。在目标1中，我们测试删除 或阻断LEAP 2将加重肥胖和葡萄糖耐受不良，我们确定 LEAP 2和ghrelin进入不同CNS区域的程度。在目标2中，我们测试在一个 严重的热量限制协议建模饥饿，删除LEAP 2将进一步提高能力， 激活GHSRs以提高血糖和存活率。在目标3中，我们选择性地从肝脏或肝脏中删除LEAP 2。 肠-LEAP 2的两个主要来源-和评估血浆LEAP 2和代谢的变化， 对长期高脂饮食暴露和严格的热量限制的反应。我们将使用四个集合 新的、未发表的重组小鼠品系，其允许我们删除或位点选择性删除LEAP 2和/或 生长素释放肽，连同诱导LEAP 2表达敲低的新型病毒载体，以及LEAP 2 中和单克隆抗体。我们的研究将提供基本的洞察力的功能意义 最近表征的GHSR拮抗剂和反向激动剂LEAP 2和相关的GHSR激动剂酰基- 生长激素释放肽在营养过剩环境下肥胖和葡萄糖耐受不良的发展中的作用， 在严重限制热量摄入的情况下发生危及生命的低血糖。

PROJECT SUMMARY The hormone acyl-ghrelin serves as a key regulator of eating, body weight, blood glucose, and survival upon binding to its receptor, GHSR. In the past project period, we studied regulation of ghrelin secretion. A major finding was that under a severe caloric restriction protocol modeling starvation, ghrelin cell-expressed 1- adrenergic receptors (1ARs) mediate ghrelin secretion, which in turn defends against marked hypoglycemia and mortality. Also, following the recent identification of liver-enriched antimicrobial peptide 2 (LEAP2) as an endogenous GHSR antagonist, we extended our studies to characterize LEAP2 function at the cellular level and to determine plasma LEAP2 changes due to various metabolic perturbations in human subjects and mice. We found that LEAP2 both hyperpolarizes and prevents acyl-ghrelin from activating arcuate NPY neurons, suggesting that LEAP2 serves as both a GHSR inverse agonist and antagonist. Also, we found that plasma LEAP2 is regulated by metabolic status: its levels increase with obesity and rising blood glucose and decrease with fasting and weight loss. These changes were mostly opposite of those of acyl-ghrelin. Collectively, this led us to propose the following model of LEAP2 function: 1) In obese states, LEAP2 rises and acyl-ghrelin falls, shifting the plasma LEAP2/acyl-ghrelin molar ratio higher and thus limiting acyl-ghrelin’s capacity to worsen obesity and glucose intolerance by raising food intake, body weight and blood glucose. 2) In nutritionally deficient states, such as that induced by severe caloric restriction, a fall in LEAP2 creates an environment in which elevated acyl-ghrelin can most effectively act to prevent life-threatening hypoglycemia. In the current R01 proposal, we test this model in 3 aims by using a combination of mostly novel tools to delete, knockdown, and/or neutralize LEAP2 in settings of obesity and severe caloric restriction. In Aim 1, we test whether deleting or blocking LEAP2 will exacerbate obesity and glucose intolerance in obesogenic settings, and we determine the extent to which LEAP2 and ghrelin gain access to different CNS regions. In Aim 2, we test whether under a severe caloric restriction protocol modeling starvation, deleting LEAP2 will further enhance the capacity of activated GHSRs to boost blood glucose and survival. In Aim 3, we delete LEAP2 selectively from liver or intestine – the two predominant sources of LEAP2 – and assess changes in plasma LEAP2 and metabolism in response to long-term high fat diet exposure and severe caloric restriction. We will use a collection of four new, unpublished recombinant mouse lines that allow us to delete or site-selectively delete LEAP2 and/or ghrelin, together with a novel viral vector that induces knockdown of LEAP2 expression, and a LEAP2 neutralizing monoclonal antibody. Our studies will provide fundamental insight into the functional significance of the recently characterized GHSR antagonist and inverse agonist LEAP2 and the related GHSR agonist acyl- ghrelin in the development of obesity and glucose intolerance under settings of nutritional overabundance and in the development of life-threatening hypoglycemia under settings of severe caloric restriction.