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

来源：
https://reporter.nih.gov/project-details/8741565
关键词：
Adipocytes Adipose tissue Agonist Bacteria Biochemical Biological Assay Brown Fat Cell Differentiation process Cell Lineage Chimeric Proteins Cloning Dexamethasone Fatty acid glycerol esters Goals In Vitro Insulin Laboratories Ligands Mass Spectrum Analysis Molecular N-terminal Nuclear Extract Nuclear Receptors Obesity PPAR gamma Pathway interactions Pattern Phase Phosphotransferases Process Proteins Regulation Role Signal Pathway Signal Transduction Pathway Stimulus Testing Tissue Expansion Transcriptional Activation adipocyte differentiation base cofactor gain of function gene induction in vivo interest loss of function novel research study response transcription factor

Adipocytes Adipose tissue Agonist Bacteria Biochemical Biological Assay Brown Fat Cell Differentiation process Cell Lineage Chimeric Proteins Cloning Dexamethasone Fatty acid glycerol esters Goals In Vitro Insulin Laboratories Ligands Mass Spectrum Analysis Molecular N-terminal Nuclear Extract Nuclear Receptors Obesity PPAR gamma Pathway interactions Pattern Phase Phosphotransferases Process Proteins Regulation Role Signal Pathway Signal Transduction Pathway Stimulus Testing Tissue Expansion Transcriptional Activation adipocyte differentiation base cofactor gain of function gene induction in vivo interest loss of function novel research study response transcription factor

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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剂。通过自然和合成激动剂激活该转录因子会导致诱导参与脂肪分化和胰岛素敏化的基因。尽管自从90年代初期发现该因素以来，已经对Ppargamma配体依赖性转录激活的机制进行了广泛的研究，但ppargamma的配体独立的功能尚未完全阐明。实验室的主要重点之一是鉴定可以调节ppargamma配体无关活性的新型分子和途径。为了识别新型的ppargamma调节剂，我们采取了以下方法： 1）我们已经分析了几个在脂肪细胞分化过程中似乎表达的辅助因子，并评估了它们充当ppargamma潜在转录辅助因子的能力； 2）我们生成了几种表达ppargamma不同领域的ppargamma-gst融合构建体。这些在细菌中产生的融合蛋白已用于生化测定中，以纯化潜在的新型N末端相互作用。已经分析了从前膜细胞获得的核提取物和完全分化的脂肪细胞，并通过质谱法鉴定出了新型相互作用蛋白。除了表征新颖的ppargamma相互作用分子外，我们还集中在新型信号通路的表征上，这些途径可以增强对地塞米松刺激的响应。为此，我们分析了在脂肪分化的不同阶段中几种激酶的表达模式，并确定了几种潜在的激酶，这些激酶可能参与掺杂脂肪生成过程的调节，特别是针对地塞米松刺激的响应。目前，我们正在进行前脂肪细胞中的功能获得实验，以评估这些激酶调节脂肪分化的能力。此外，我们还通过专门在脂肪组织中进行的功能丧失实验来测试这些激酶在体内的作用。该项目的第三个重点是鉴定了控制脂肪细胞分化早期阶段的新因素，该因素是脂肪细胞分化的早期阶段。我们目前正在测试几种新候选转录因子的作用，作为脂肪细胞谱系的早期决定因素。