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Identification of novel modulators of adipocyte differentiation

脂肪细胞分化的新型调节剂的鉴定

基本信息

批准号：
8741565
负责人：
Elisabetta Mueller
金额：
$ 49.41万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

The nuclear receptor PPARgamma is considered the central regulator of adipocyte differentiation. Activation of this transcription factor via natural and synthetic agonists leads to the induction of genes involved in fat differentiation and insulin sensitization. Although the mechanisms of PPARgamma's ligand-dependent transcriptional activation have been studied extensively since the discovery of this factor in the early nineties, the ligand-independent function of PPARgamma has not yet been fully elucidated. One of the main focus of the lab is the identification of novel molecules and pathways that can modulate PPARgamma's ligand-independent activity. In order to identify novel PPARgamma modulators we have taken the following approaches: 1) We have analyzed several cofactors candidates that appear to be expressed during adipocyte differentiation and assessed their ability to function as potential transcriptional cofactors for PPARgamma; 2) We have generated several PPARgamma-GST fusion constructs that express distinct domains of PPARgamma. These fusion proteins produced in bacteria have been utilized in biochemical assays to purify potential novel N-terminal interactors. Nuclear extracts obtained from preadipocytes and fully differentiated adipocytes have been analyzed and novel interacting proteins have been identified by mass spectrometry. In addition to the characterization of novel PPARgamma-interacting molecules, we are focusing on the characterization of novel signaling pathways that can enhance differentiation in response to dexamethasone stimuli. For this purpose we have analyzed the pattern of expression of several kinases during different phases of fat differentiation and have identified several potential kinases that could be involved in the regulation of the adipogenic process specifically in response to dexamethasone stimulation. We are currently performing gain-of-function experiments in preadipocytes to assess the ability of these kinases to modulate fat differentiation. In addition we are testing the role of these kinases in vivo, via loss-of-function experiments carried out specifically in adipose tissue. The third focus of this project is the identification of novel factors that control early phases of adipocyte differentiation that precede PPARgamma expression. We are currently testing the role of several new candidate transcription factors as early determinants of the adipocyte cell lineage.

核受体PPARgamma被认为是脂肪细胞分化的中心调节因子。这种转录因子通过天然和合成激动剂的激活导致参与脂肪分化和胰岛素敏化的基因的诱导。尽管自九十年代早期发现该因子以来，已经广泛研究了PPARgamma的配体依赖性转录激活机制，但PPARgamma的配体非依赖性功能尚未完全阐明。该实验室的主要重点之一是鉴定可以调节PPARgamma的配体非依赖性活性的新分子和途径。为了鉴定新的PPARgamma调节剂，我们采取了以下方法： 1)我们已经分析了几种在脂肪细胞分化过程中表达的候选辅因子，并评估了它们作为潜在的PPARgamma转录辅因子的功能; 2)我们已经产生了几个PPARgamma-GST融合构建体，表达不同的结构域的PPARgamma。这些在细菌中产生的融合蛋白已被用于生物化学测定以纯化潜在的新型N-末端相互作用物。从前脂肪细胞和完全分化的脂肪细胞中获得的核提取物已被分析，并通过质谱鉴定了新的相互作用蛋白。除了新的PPARgamma相互作用分子的特性，我们正在集中在新的信号通路，可以增强响应地塞米松刺激分化的特性。为此，我们分析了几种激酶在脂肪分化的不同阶段的表达模式，并确定了几种可能参与调节脂肪形成过程的潜在激酶，特别是对地塞米松刺激的反应。我们目前正在前脂肪细胞中进行功能获得实验，以评估这些激酶调节脂肪分化的能力。此外，我们正在测试这些激酶在体内的作用，通过功能丧失的实验进行专门在脂肪组织。该项目的第三个重点是识别控制PPARgamma表达前脂肪细胞分化早期阶段的新因子。我们目前正在测试的作用，几个新的候选转录因子作为脂肪细胞系的早期决定因素。