Cross-talk between Leptin and Estrogen Signaling in Hypothalamic Arcuate Neurons

描述（由申请人提供）：本项目的主要目标是阐明kisspeptin神经元中瘦素和17-雌二醇信号传导之间的串扰。先天性瘦素缺乏和/或由于瘦素突变导致的瘦素功能丧失可导致肥胖和促性腺功能低下。下丘脑性腺功能减退及其相关障碍可通过瘦素治疗逆转。瘦素通过其同源受体，瘦素受体（LRs）发出信号。长异构体（LRb）是该受体的主要信号形式，在下丘脑神经元中大量表达，包括弓状促性腺激素释放激素（GnRH）神经元，但在下丘脑神经元中不表达。因此，瘦素对GnRH神经元的影响被认为是通过神经元与GnRH神经元的突触间接介导的。下丘脑kisspeptin神经元在调节GnRH释放从而控制生殖中起关键作用。此外，KiSS1 mRNA在肥胖和不育ob/ob小鼠中降低，并且在给药瘦素后KiSS1 mRNA水平升高。此外，KiSS1 mRNA和kisspeptin蛋白受到17-雌二醇（E2）的高度调控，最近，我们发现E2对雌性豚鼠弓形kisspeptin神经元有差异调控，在负反馈时抑制表达，在正反馈时增强表达。此外，我们发现瘦素通过一种新的信号通路使POMC神经元去极化，该通路与典型瞬时受体电位（TRPC）通道的激活相耦合，kisspeptin神经元可能受到类似的调节。因此，我们目前的工作重点是下丘脑弓状kisspeptin神经元以及E2和瘦素之间的相互作用，通过多种信号级联作用影响kisspeptin神经元的兴奋性并最终影响生殖周期。我们的多学科方法结合了一系列独特的细胞和分子工具以及我们的综合专业知识（电生理学，分子生物学，组织化学和全动物生理学）。我们的工作假设是kisspeptin神经元是雌性GnRH神经元兴奋驱动的“守门者”，E2和瘦素在这些神经元中复杂的相互作用控制着进食和禁食状态下的排卵周期。因此，我们的具体目的如下：(1)研究去卵巢雌性豚鼠弓形kisspeptin神经元中瘦素信号通路的特征。(2)研究E2在正反馈和负反馈条件下对弓状kisspeptin神经元的影响。(3)探讨E2在禁食与进食豚鼠正反馈过程中对弓状kisspeptin神经元的作用。(4)探讨E2和禁食对kisspeptin神经元K-ATP通道表达的影响。了解瘦素和E2信号在弓形kisspeptin神经元中的收敛性，将有助于深入了解这些激素在向生殖轴传递代谢信号方面的基本作用。

DESCRIPTION (provided by applicant): The primary goal of this project is to elucidate the cross-talk between leptin and 17-estradiol signaling in kisspeptin neurons. Congenital leptin deficiency and/or loss of leptin function due to mutations in leptin can cause obesity and hypogonadotropic hypogonadism. Hypothalamic hypogonadism and its associated disturbances can be reversed by administration of leptin. Leptin signals via its cognate receptors, leptin receptors (LRs). The long isoform (LRb) is the predominant signaling form of the receptor and is abundantly expressed in hypothalamic neurons, including arcuate proopiomelanocortin (POMC) and kisspeptin neurons, but not in gonadotropin releasing-hormone (GnRH) neurons. Therefore, the effects of leptin on GnRH neurons are thought to be mediated indirectly via neurons synapsing on GnRH neurons. Hypothalamic kisspeptin neurons play a critical role in modulating GnRH release and hence the control of reproduction. Moreover, KiSS1 mRNA is reduced in obese and infertile ob/ob mice, and the levels of Kiss1 mRNA increase after administration of leptin. Furthermore, KiSS1 mRNA and kisspeptin protein are highly regulated by 17- estradiol (E2), and recently, we have found that E2 differentially regulates arcuate kisspeptin neurons in the female guinea pig, inhibiting expression during negative feedback and augmenting expression during positive feedback. In addition, we have discovered that leptin depolarizes POMC neurons via a novel signaling pathway that is coupled to activation of canonical transient receptor potential (TRPC) channels, and kisspeptin neurons may be similarly regulated. Therefore, our current work focuses on hypothalamic arcuate kisspeptin neurons and the interaction between E2 and leptin acting through multiple signaling cascades to affect kisspeptin neuronal excitability and ultimately the reproductive cycle. Our multidisciplinary approach incorporates a unique array of cellular and molecular tools and our combined expertise (electrophysiology, molecular biology, histochemistry and whole animal physiology). Our working hypothesis is that the kisspeptin neurons are the "gate-keeper" of excitatory drive to GnRH neurons in the female, and it is the complex interaction of E2 and leptin in these neurons that control the ovulatory cycle in fed and fasted states. Therefore, our specific aims are the following: (1) To characterize the leptin signaling pathway in arcuate kisspeptin neurons in ovariectomized female guinea pigs. (2) To characterize the effects of E2 on arcuate kisspeptin neurons during positive feedback versus negative feedback. (3) To elucidate the actions of E2 on arcuate kisspeptin neurons during positive feedback in fasted versus fed guinea pigs. (4) To elucidate the effects of E2 and fasting on the expression of K-ATP channels in kisspeptin neurons. Understanding the convergence of leptin and E2 signaling in arcuate kisspeptin neurons will provide insight into the fundamental role of these hormones in conveying metabolic cues to the reproductive axis. PUBLIC HEALTH RELEVANCE: Excessive exercise, eating disorders or weight loss in women can cause amenorrhea, which is characterized by low levels of serum 17-estradiol and anovulation. It is known that during negative energy balance the hormone leptin that is released from fat stores is drastically reduced and may contribute to hypothalamic amenorrhea conditions. However, leptin receptors are not expressed in hypothalamic gonadotropin releasing hormone (GnRH) neurons, which are directly responsible for the control of the ovulatory cycle, but in kisspeptin neurons that are essential for the excitatory input to GnRH neurons. Therefore, the current studies will characterize the cross-talk between leptin and 17-estradiol signaling in kisspeptin neurons in fed and fasted states. Understanding the convergence of leptin and estrogen signaling in arcuate kisspeptin neurons will provide insight into the fundamental role of these hormones in conveying metabolic cues to the reproductive axis.