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）：该修订项目的远程目标是了解脂肪细胞中调节胰岛素敏感性和代谢通量的主要分子机制。小鼠脂肪细胞葡萄糖代谢的破坏会引起肌肉胰岛素耐药性和糖尿病，同时增加脂肪细胞中的葡萄糖利用率可增强全身葡萄糖耐受性。因此，脂肪细胞代谢的新基因和调节因子的识别和详细表征是该领域的基本目标。我们使用高通量RNAi筛选获得的令人兴奋的初步数据揭示了两种蛋白质是脂肪细胞中葡萄糖和脂肪酸氧化的新型全球调节剂-Corepressor RIP140和蛋白激酶MAP4K4。我们的数据表明，RIP140和MAP4K4的作用在PPAR-？中融合了，这些蛋白质对这些蛋白质负调节，这些作用可能是高度整合的。在培养的脂肪细胞中将RIP140或MAP4K4沉默：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，并增强3.）增强酶的表达，从而可以控制线粒体生物发生和氧气消耗。在缺乏RIP140的脂肪细胞中观察到氧化代谢明显增加。令人惊讶的是，RIP140 KO小鼠在喂养高脂饮食时对肥胖症和葡萄糖不耐症具有抗性。我们在这里建议确定RIP140和MAP4K4抑制和TZD调控的基因集，以检验PPAR的假设？是他们监管的目标。潜在PPAR的两个机制？将评估抑制作用：1。）PPAR之间可能的直接相互作用？和RIP140或MAP4K4，以及2。）PPAR的直接磷酸化？还是PPAR的监管机构？例如RIP140本身由MAP4K4本身。将对PPAR？ - 敏感和不敏感基因的启动子区域进行芯片分析，以确定RIP140或MAP4K4是否存在于PPAR中？在这些启动子。实验RIP140和MAP4K4对PPAR的探测分子机制？现在提出了使用确认的基于TZD敏感的荧光素酶的PPAR的记者？脂肪细胞中的启动子活性。质谱法将在RIP140，PPAR？和MAP4K4的其他潜在底物上识别可能的磷酸化位点，然后在转录调控中进行功能分析。我们还将测试TZD对PPAR的分子基础？因为它与MAP4K4和RIP140功能有关。最后，新提出的研究探讨了RIP140的代谢作用与小鼠的葡萄糖耐量如何相关。我们将检验以下假设：RIP140 KO小鼠中脂肪酸和肌肉中脂肪酸的氧化大大增强，可以释放出脂肪酸衍生物对胰岛素信号传导的抑制作用。这些研究将揭示这些新型脂肪细胞代谢和功能的全球调节剂的基本作用机理。