The Agouti Related Protein and its Role in Human Obesity

描述(申请人提供)：AGRP在肥胖和糖尿病小鼠中表达上调，当在转基因小鼠中过度表达时，会刺激过度吞噬和肥胖的发展。人类同源基因hAGRP已经被分离出来，并具有类似的分子和生理特性，在弓状核和外周分别表达一种转录本。我们最近确定了人AGRP基因的基因结构，并确定了其最小启动子。确定了转录因子的多个结合位点，包括可能潜在地介导瘦素在下丘脑中的作用的STAT反式激活因子的假定结合位点。5‘非编码外显子仅在外周细胞系中具有显著的启动子活性，提示其在外周特异性转录物的表达中起作用。我们还鉴定了启动子中的一种多态性，它对启动子活性和结合转录因子的亲和力有显著影响，这在下丘脑和外周来源的细胞系中进行了测试。在两个不同的队列中，具有高启动子活性的基因与肥胖和2型糖尿病(T2 DM)显著相关。此外，该基因编码区的多态与低体重指数和低腹肥胖显著相关，但仅在老年人中相关，这表明hAGRP在预防晚期肥胖方面起到了作用。我们假设，由hAGRP启动子决定的下丘脑和外周hAGRP的数量增加将导致吞噬功能亢进和肥胖的发展。因此，我们建议完全表征hAGRP的启动子，并确定调控该基因表达的转录因子和序列基序(特定目标1)。我们还将确定激素/燃料底物对AGRP启动子活性和内源性表达的影响，并确定该基因启动子中的激素/底物反应元件(特定目标2)。此外，我们将研究hAGRP多态与肥胖和T2 DM的关系，而AGRP的系统水平将与启动子多态以及激素/燃料底物的系统水平相关(特定目标3)。鉴于hAGRP是一种强大的食欲效应因子，重要的是我们要确定其表达的调控机制，并确定其在食物摄入和人类肥胖发展中的作用。

DESCRIPTION (provided by applicant): The Agouti Related Protein (AGRP) is upregulated in obese and diabetic mice and stimulates hyperphagia and the development of obesity when overexpressed in transgenic mice. The human ortholog, hAGRP, has been isolated and has similar molecular and physiological properties, expressing one transcript in the arcuate nucleus and one in the periphery. We recently determined the gene structure and identified the minimal promoter of the human AGRP gene. Multiple binding sites for transcription factors were identified including putative binding sites for the STAT transactivators that may potentially mediate leptin's action in the hypothalamus. The 5' non-coding exon had significant promoter activity in a periphery cell line only, suggesting its role in the expression of the periphery-specific transcript. We also identified a polymorphism in the promoter that had significant impact on promoter activity and affinity to bind transcription factors, as tested in hypothalamus- and periphery-derived cell lines. The genotype with the high promoter activity was significantly associated with obesity and Type 2 Diabetes Mellitus (T2DM) in two different cohorts. Moreover, a polymorphism in the coding region of the gene was significantly associated with low body mass index and low abdominal adiposity but in older people only, suggesting a role for hAGRP in preventing late on set obesity. We hypothesize that increased amounts of hAGRP in the hypothalamus and the periphery, as determined by its promoter, will result in hyperphagia and the development of obesity. We therefore propose to completely characterize the promoter of hAGRP and identify the transcription factors and sequence motifs that regulate expression of the gene (Specific Aim 1). We will also determine the impact of hormones/fuel substrates on promoter activity and endogenous expression of AGRP, and identify the hormone/substrate response elements in the promoter of the gene (Specific Aim 2). Furthermore, we will investigate the association of hAGRP polymorphisms with obesity and T2DM, while the systemic levels of AGRP will be correlated with promoter polymorphisms as well as with the systemic levels of hormones/fuel substrates (Specific Aim 3). Given that hAGRP is a potent appetite effector, it is important that we identify the regulatory mechanisms for its expression and determine its role in food intake and the development of human obesity.