Transcriptional Control of Obesity

虽然已有20多个神经元表达的基因参与了体重的调节，但这些基因在能量平衡变化时的转录和转录后表达机制仍不清楚。最近来自我的实验室的证据表明，NhIh2转录因子在控制体重的下丘脑基因的调控中发挥着关键作用。NhIh2基因敲除小鼠(N2KO)表现出成人型肥胖，NhIh2在控制食欲的下丘脑区域--包括下丘脑的弓状核(ARC)--表达。在双标记原位杂交研究中，我们发现ARC内几乎所有的前阿片黑素皮质素(POMC)神经元都表达NhIh2。在这些神经元中，POMC前体蛋白被加工成α-黑素细胞刺激素(AlphaMSH)，这是一种调节食物摄入量和能量消耗的多肽。在比较N2KO和正常小鼠的POMC mRNA和多肽水平的研究中，我们发现，虽然两组POMC mRNA的水平相同，但N2KO动物的两种加工形式POMC的水平显著降低，而α-MSH水平略有下降。这些发现表明，N2KO动物可能存在减少POMC前体加工的缺陷。事实上，在N2KO动物中，将POMC分解为生物活性多肽前激素转换酶I(PC1)和II(PC2)所必需的两种加工酶的mRNA水平降低了80%。因此，PC1和PC2很可能是NhIh2转录因子的直接基因调控靶点，完全加工神经肽的减少导致N2KO动物肥胖。此外，我们预测NhIh2是瘦素信号通路的直接靶点，因为它在含有瘦素受体的POMC神经元中表达，并且在其启动子中含有STAT基序。基于这些初步发现，我们认为正能量平衡增加了NhIh2的表达，进而提高了PC1和PC2基因的转录速率。为了验证这一假设，我们将在前两个特定目标中询问，N2KO动物的NhIh2、PC1和PC2是否因正能量平衡而增加，以及PC1和PC2基因是否丢失。在具体目标3中，我们将切换到对NhIh2启动子的分子研究，以确定AP1和STAT3转录因子是否可以与NhIh2基因结合并反式激活。然后我们将在特定的目标4中询问NhIh2转录因子是否与激活的STAT3结合并反式激活PC1启动子。拟议的实验使用分子和基于动物的方法来提出一个关于转录和转录后控制体重的机制的基本问题。

Although over 20 neuronally-expressed genes have been implicated in the regulation of body weight, the transcriptional and post-transcriptional mechanisms through which these genes are expressed in response to changes in energy balance remain unclear. Recent evidence from my laboratory suggests that the NhIh2 transcription factor plays a key role in the regulation of hypothalamic genes controlling body weight. NhIh2 knockout mice (N2KO) display adult-onset obesity and NhIh2 is expressed in the hypothalamic regions that control appetite-including the arcuate ncuelus (ARC) of the hypothalamus. In dual-label in situ hybridization studies, we found that nearly all pro- opiomelanocortin (POMC) neurons in the ARC expressed NhIh2. In these neurons, the POMC precursor protein is processed to alpha- melanocyte stimulating hormone (alphaMSH), a peptide that regulates food intake and energy expenditure. In studies comparing POMC mRNA and peptide levels in N2KO and normal mice, we found that while POMC mRNA levels were identical between the two groups, levels of two processed forms of POMC, beta-endorphin (betaEND) and adrenocorticotropin hormone (ACTH) were dramatically reduced, while levels of alphaMSH were modestly reduced in the N2KO animals. These findings suggest that N2KO animals could have a defect that reduces processing of the POMC precursor. Indeed, mRNA levels for two of the processing enzymes necessary for cleaving POMC into bioactive peptides prohormone convertases I (PC1) and II (PC2) were reduced up to 80 percent in N2KO animals. Thus, it is likely that PC1 and PC2 are direct gene regulatory targets of the NhIh2 transcription factor and that a reduction in fully processed neuropeptides contributes to obesity in the N2KO animals. In addition, we predict that NhIh2 is a direct target of leptin-signaling pathways, as it is expressed in the leptin-receptor containing POMC neurons and contains STAT motifs in its promoter. Based on these preliminary findings, we propose that positive energy balance increases the expression of NhIh2, which in turn increases the transcription rate of the PC1 and PC2 genes. To test this hypothesis, we will ask in the first two specific aims, if NhIh2, PC1 and PC2 are increased in response to a positive energy balance and if the of the PC1 and PC2 genes is lost in N2KO animals. In specific aim 3, we will switch to a molecular investigation of the NhIh2 promoter to determine if AP1 and STAT3 transcription factors can bind to and transactivate the NhIh2 gene. We will then ask in specific aim 4 if the NhIh2 transcription factor, in cooperation with activated STAT3, binds to and transactivates the PC1 promoter. The proposed experiments use both molecular and animal-based approaches to ask a fundamental question about transcriptional and post- transcriptional mechanisms controlling body weight.