Insulin Signaling and Metabolic Regulation in Adipocytes

脂肪细胞中的胰岛素信号传导和代谢调节

• 批准号: 7996752
• 负责人: MICHAEL P CZECH
• 金额: $ 19.83万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-14 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7996752
• 关键词: 2,4-thiazolidinedione; Ablation; Address; Adipocytes; Adipose tissue; Biogenesis; Biological Assay; Carbohydrates; Cell Respiration; Chimeric Proteins; Co-Immunoprecipitations; Collaborations; Complex; Data; Diabetes Mellitus; Diet; Enzymes; Exhibits; Fatty Acids; Fatty acid glycerol esters; GLUT4 gene; Gene Expression; Genes; Glucose; Glucose Intolerance; Goals; Infusion procedures; Insulin; Insulin Resistance; Knockout Mice; Laboratories; Link; Lipolysis; Luciferases; MAP Kinase Gene; MAP4K4 gene; Mass Spectrum Analysis; Measures; Mediating; Metabolic; Metabolism; Mitochondria; Molecular; Molecular Probes; Mus; Muscle; NRIP1 gene; Obesity; Oxygen Consumption; Pathway interactions; Peptide Mapping; Peroxisome Proliferator-Activated Receptors; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Principal Investigator; Process; Promoter Regions; Protein Kinase; Proteins; RNA Interference; Regulation; Regulator Genes; Reporter; Repression; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Serum; Site; Testing; Thiazolidinediones; Transcriptional Regulation; Triglycerides; attenuation; base; blood glucose regulation; design; fatty acid metabolism; fatty acid oxidation; feeding; genetic regulatory protein; glucose metabolism; glucose tolerance; glucose uptake; human NRIP1 protein; insulin sensitivity; insulin signaling; knockout gene; lipid metabolism; mutant; novel; programs; promoter; protein function; research study; response; sterol esterase; transcription factor

DESCRIPTION (provided by applicant): The long-range goal of this revised project is to understand major molecular mechanisms that regulate insulin-sensitivity and metabolic flux in adipocytes. Disruptions in adipocyte glucose metabolism in mice can cause muscle insulin resistance and diabetes, while augmenting glucose utilization in adipocytes enhances whole body glucose tolerance. Thus, the identification and detailed characterization of novel genes and regulators of adipocyte metabolism are fundamental objectives in this field. Exciting preliminary data we obtained using high-throughput RNAi-based screens have revealed two proteins as novel global regulators of glucose and fatty acid oxidation in adipocytes - the corepressor RIP140 and the protein kinase MAP4K4. Our data suggest the hypothesis that the actions of both RIP140 and MAP4K4 converge at PPAR-?, which is negatively regulated by these proteins, and these actions may be highly integrated. Silencing either RIP140 or MAP4K4 in cultured adipocytes: 1.) enhances expression of enzymes in carbohydrate and fatty acid oxidation, 2.) enhances insulin-sensitive glucose uptake, and 3.) enhances expression of enzymes that control mitochondrial biogenesis and oxygen consumption. Markedly increased oxidative metabolism is observed in adipocytes lacking RIP140. Strikingly, RIP140 KO mice are resistant both to obesity and to glucose intolerance when fed a high fat diet. We propose here to identify the set of genes that is common to RIP140 and MAP4K4 repression and TZD regulation to test the hypothesis that PPAR? is a target of their regulation. Two mechanisms of potential PPAR? repression will be evaluated: 1.) possible direct interactions between PPAR? and RIP140 or MAP4K4, and, 2.) direct phosphorylation of PPAR? or regulators of PPAR? such as RIP140 itself by MAP4K4. ChIP analysis of promoter regions of PPAR?-sensitive and insensitive genes will be performed to determine whether RIP140 or MAP4K4 are present in complexes with PPAR? at these promoters. Experiments to probe molecular mechanisms of RIP140 and MAP4K4 actions on PPAR? are now proposed, using a confirmed TZD-sensitive luciferase-based reporter of PPAR? promoter activity in adipocytes. Mass spectrometry will identify possible phosphorylation sites on RIP140, PPAR?, and other potential substrates of MAP4K4, followed by their functional analysis in transcriptional regulation. We shall also test the molecular basis of TZD action on PPAR? as it relates to MAP4K4 and RIP140 functions. Finally, newly proposed studies address how the metabolic actions of RIP140 are linked to glucose tolerance in mice. We shall test the hypothesis that greatly enhanced fatty acid oxidation in fat and perhaps muscle in RIP140 KO mice releases the inhibition by fatty acid derivatives on insulin signaling. These studies will reveal underlying mechanisms of action of these novel global regulators of adipocyte metabolism and function.

描述（由申请人提供）：该修订项目的长期目标是了解调节脂肪细胞中胰岛素敏感性和代谢流的主要分子机制。小鼠脂肪细胞葡萄糖代谢的破坏可导致肌肉胰岛素抵抗和糖尿病，而增加脂肪细胞中的葡萄糖利用可增强全身葡萄糖耐量。因此，脂肪细胞代谢的新基因和调节因子的鉴定和详细表征是该领域的基本目标。我们使用基于高通量 RNAi 的筛选获得了令人兴奋的初步数据，揭示了两种蛋白质作为脂肪细胞中葡萄糖和脂肪酸氧化的新型全局调节剂 - 辅阻遏物 RIP140 和蛋白激酶 MAP4K4。我们的数据表明，RIP140 和 MAP4K4 的作用均集中于 PPAR-α，而 PPAR-α 受到这些蛋白质的负调控，并且这些作用可能是高度整合的。在培养的脂肪细胞中沉默 RIP140 或 MAP4K4：1.) 增强碳水化合物和脂肪酸氧化中酶的表达，2.) 增强胰岛素敏感的葡萄糖摄取，以及 3.) 增强控制线粒体生物发生和耗氧的酶的表达。在缺乏 RIP140 的脂肪细胞中观察到氧化代谢显着增加。引人注目的是，当喂食高脂肪饮食时，RIP140 KO 小鼠能够抵抗肥胖和葡萄糖不耐受。我们在此建议鉴定 RIP140 和 MAP4K4 抑制以及 TZD 调节所共有的一组基因，以检验 PPAR？是他们监管的一个目标。潜在 PPAR 的两种机制？将评估抑制：1.) PPAR 之间可能存在直接相互作用？和 RIP140 或 MAP4K4，以及，2.) PPAR 的直接磷酸化？或PPAR的调节剂？例如 MAP4K4 的 RIP140 本身。对 PPAR? 敏感和不敏感基因的启动子区域进行 ChIP 分析，以确定 RIP140 或 MAP4K4 是否存在于与 PPAR? 的复合物中。在这些发起人身上。探究 RIP140 和 MAP4K4 对 PPAR 作用的分子机制的实验？现在建议使用经过证实的 TZD 敏感的基于荧光素酶的 PPAR 报告基因？脂肪细胞中的启动子活性。质谱分析将鉴定 RIP140、PPAR? 和 MAP4K4 其他潜在底物上可能的磷酸化位点，然后对其在转录调控中进行功能分析。我们还将测试 TZD 对 PPAR 作用的分子基础？因为它与 MAP4K4 和 RIP140 功能相关。最后，新提出的研究探讨了 RIP140 的代谢作用如何与小鼠的葡萄糖耐量相关。我们将检验这样的假设：RIP140 KO 小鼠的脂肪和肌肉中的脂肪酸氧化大大增强，从而释放了脂肪酸衍生物对胰岛素信号传导的抑制。这些研究将揭示这些新型脂肪细胞代谢和功能全局调节剂的潜在作用机制。