Physiological role of PRDM16 in brown fat development and energy balance

PRDM16在棕色脂肪发育和能量平衡中的生理作用

• 批准号: 8133390
• 负责人: Patrick Seale
• 金额: $ 24.65万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-05 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8133390
• 关键词: Acetyltransferase; Acute; Adipocytes; Adipose tissue; Adult; Alleles; Animals; Appearance; Applications Grants; Autologous; Autologous Transplantation; Back; Binding; Biochemical; Biological Assay; Blood Vessels; Boston; Breeding; Brown Fat; C-Terminal Binding Protein 1; Calories; Catecholamines; Cell Culture Techniques; Cell Differentiation process; Cell Transplantation; Cells; Chemicals; Chronic; Cleft Palate; Clone Cells; Collaborations; Commit; Complex; Control Animal; Cues; Cyclic AMP; DNA; Data; Defect; Degenerative polyarthritis; Deposition; Development; Diet; Disease; EP300 gene; ES Cell Line; EVI1 gene; Ectopic Expression; Energy Intake; Energy Metabolism; Engineering; Enhancers; Exhibits; Fatty acid glycerol esters; Fibroblasts; Food; Gene Expression; Genes; Genetic; Germ Lines; Goals; Gold; Grant; Hand; Heart Diseases; Heating; Hospitals; Hypertension; Implant; In Vitro; Inbred Strain; Insulin Resistance; Knock-out; Laboratories; Length; Leukocytes; Ligand Binding Domain; Ligands; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mentors; Metabolic; Metabolism; Mus; Muscle; Muscle Fibers; Myoblasts; Myogenin; N-terminal; NIH 3T3 Cells; Non-Insulin-Dependent Diabetes Mellitus; Obese Mice; Obesity; Pathology; Pathway interactions; Pattern; Perinatal; Phase; Phenotype; Physiological; Physiology; Positron-Emission Tomography; Prevalence; Proliferating; Protein Binding Domain; Protein Family; Proteins; Protocols documentation; Public Health; Reagent; Reporting; Repression; Resources; Risk Factors; Role; Skeletal Muscle; Skeletal Myoblasts; Specific qualifier value; Stroke; Testing; Time; Tissue Differentiation; Tissues; To specify; Transcript; Transcription Coactivator; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Transplantation; Weight Gain; Woman; Work; Zinc Fingers; adipocyte differentiation; base; cohort; design; energy balance; fetal; fighting; implantation; in vivo; lipid biosynthesis; male; member; mutant; myogenesis; novel therapeutics; precursor cell; programs; research study; response; satellite cell; skeletal; small hairpin RNA; subcutaneous; success; therapeutic target; transmission process; Acetyltransferase; Acute; Adipocytes; Adipose tissue; Adult; Alleles; Animals; Appearance; Applications Grants; Autologous; Autologous Transplantation; Back; Binding; Biochemical; Biological Assay; Blood Vessels; Boston; Breeding; Brown Fat; C-Terminal Binding Protein 1; Calories; Catecholamines; Cell Culture Techniques; Cell Differentiation process; Cell Transplantation; Cells; Chemicals; Chronic; Cleft Palate; Clone Cells; Collaborations; Commit; Complex; Control Animal; Cues; Cyclic AMP; DNA; Data; Defect; Degenerative polyarthritis; Deposition; Development; Diet; Disease; EP300 gene; ES Cell Line; EVI1 gene; Ectopic Expression; Energy Intake; Energy Metabolism; Engineering; Enhancers; Exhibits; Fatty acid glycerol esters; Fibroblasts; Food; Gene Expression; Genes; Genetic; Germ Lines; Goals; Gold; Grant; Hand; Heart Diseases; Heating; Hospitals; Hypertension; Implant; In Vitro; Inbred Strain; Insulin Resistance; Knock-out; Laboratories; Length; Leukocytes; Ligand Binding Domain; Ligands; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mentors; Metabolic; Metabolism; Mus; Muscle; Muscle Fibers; Myoblasts; Myogenin; N-terminal; NIH 3T3 Cells; Non-Insulin-Dependent Diabetes Mellitus; Obese Mice; Obesity; Pathology; Pathway interactions; Pattern; Perinatal; Phase; Phenotype; Physiological; Physiology; Positron-Emission Tomography; Prevalence; Proliferating; Protein Binding Domain; Protein Family; Proteins; Protocols documentation; Public Health; Reagent; Reporting; Repression; Resources; Risk Factors; Role; Skeletal Muscle; Skeletal Myoblasts; Specific qualifier value; Stroke; Testing; Time; Tissue Differentiation; Tissues; To specify; Transcript; Transcription Coactivator; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Transplantation; Weight Gain; Woman; Work; Zinc Fingers; adipocyte differentiation; base; cohort; design; energy balance; fetal; fighting; implantation; in vivo; lipid biosynthesis; male; member; mutant; myogenesis; novel therapeutics; precursor cell; programs; research study; response; satellite cell; skeletal; small hairpin RNA; subcutaneous; success; therapeutic target; transmission process

Aim 1: We test the hypothesis that PRDM16 can drive the formation of functional brown adipocytes in white fat depots of mice to raise energy expenditure and protect against obesity and cold exposure. Diet-Induced Obesity (DIP). The first cohort of male aP2-PRDM16 and littermate control animals are currently in week 6 of a 16 week long study in which they are consuming a high fat containing diet (55% calories from fat). During the past year, out of necessity, we back-crossed the transgenic mouse lines into the C57Biack6 genetic background. The transgenic mice were generated in the FVB background and this inbred strain is not prone to high fat diet-induced obesity. We did not have any success generating transgenic lines directly in the C57Black6 strain. C57Black6 mice are the gold-standard for the types of metabolic experiments proposed. Cold-exposure. Acute and chronic cold exposure experiments will be performed in separate cohorts of transgenic and wildtype littermates in the C57Black6 genetic background. Transplantation of PRDM16 expressing fibroblasts. The goal is to develop an autologous transplantation protocol using PRDM16-expressing precursor cells to form ectopic deposits of functional brown adipose tissue (BAT) in animals. The hypothesis is that such "synthetic" BAT would safely and effectively raise energy expenditure to reduce obesity. In new data, PRDM16 expression in stromal vascular (SV) cells from white fat promoted adipogenesis in vivo after subcutaneous transplantion, however the transplants were always small (1-2 mm), had the unilocular appearance of white adipose and did not express the thermogenic marker, UCP1. In addition, skeletal myoblasts expressing PRDM16 formed ectopic depots of adipose tissue after subcutaneous transplantation. This result demonstrates that the adipogenic action of PRDIVI16 in committed myoblastic cells is compatible with the "real" environmental cues that control adipocyte differentiation in vivo. At the same time, mechanistic studies led by Shingo Kajimura demonstrated that PRDM16 binds and strongly coactivates c/EBPp (1). c/EBP(3 is well-expressed in myogenic precursor cells and is genetically required for PRDM16-driven brown adipogenic determination. Notably, BAT from c/EBPp-deficient fetal mice displayed dramatically reduced expression of thermogenic genes and elevated levels of muscle-specific transcripts analogous to what was observed in P/?OM) 6-deficient tissue. Co-expression of PRDM16 and c/EBPp in naive fibroblasts activates both the adipogenic and thermogenic gene program that are unique to brown adipocytes. Furthermore, these fibroblasts gave rise to multilocular, UCP1-expressing and ^^FDG-PET positive BAT in mice six weeks after subcutaneous implantation. These results indicate that a transcriptional unit containing c/EBPp and PRDM16 drives the brown adipose phenotype. Aim 2: We investigate the physiological requirement for PRDI\/I16 in BAT and whole body metabolism by creating and characterizing tissue-specific PRD/Wt6-deficient mice. We have now succeeded in generating 7 high percentage agouti chimeric mice from PRDMId"""^"^'* ES cell lines (4 independent cell clones). These animals are currently breeding with wildtype mice to establish germ-line transmission of the targeted allele. After the grant proposal was submitted, we became aware of PRD/W^6-deficient mice that were generated in the laboratory of David R. Beier at Brigham & Women's Hospital, Boston. The knock-out animals are perinatal lethal and have a pronounced cleft palate (B. Bjork, D. Beier et a/., in review). In collaboration with the Beier lab, we have analyzed the BAT phenotype from fetal PRD/W76-deficient mice. As now reported, Pf?DM^6-deficient BAT contains large lipid droplets and a dramatic reduction in the expression of thermogenic genes (2). This result reveals a genetic requirement for PRDM16 in the normal development of the tissue. In addition, the PRDM16^' BAT expressed higher levels of skeletal muscle (Sl\/l)-specific genes (2) supporting the notion that SIVl and BAT may arise from a common lineage (3). Although PRDA/f76-deficient BAT exhibits a dysregulated pattern of gene expression, it retains significant brown adipose attributes and is still recognizable as BAT. Therefore, the chronic loss of PRDM16 in vivo does not cause a total loss of BAT differentiation. This is different from the shRNA-based cell culture knockdown studies in which depletion of PRDIVlie in primary brown adipogenic precursors blocked brown adipogenesis and promoted overt skeletal myogenesis (2). We therefore hypothesize that another closely related member of the PR-domain containing family of proteins may partially compensate for the chronic loss of PRDM16 in BAT development. Indeed, PRDM3 and PRDM16 are closely related by sequence comparisons and share significant sequence similarity especially within the two conserved zinc-finger DNA/protein binding domains. We have therefore tested in our cell culture assays whether PRDM3 functions in brown adipogenesis. Preliminary studies show that PRDM3 is a key regulator of PPARY2 and adipogenic differentiation. Ectopic expression of PRDM3 in non-adipogenic fibroblasts such as NIH-3T3 cells stimulates adipogenesis including induction of some BAT-related genes in a cAMP dependent manner like UCP1 and PGC-1a. Aim 3: We investigate the mechanistic basis for PRDM16 function in: (1) activating adipogenesis via PPARy and (2) repressing myogenesis via binding to CtBP1/2.- Activation of PPARs. We have now reported that PRDIVI16 is a strong and ligand-dependent co-activator of both PPARy and PPARa (2). Moreover, agonism of PPARy was necessary for the adipogenic action of PRDM16 in cultured myoblasts. In vitro binding experiments revealed that PRDIVI16 is only able to bind to the full-length PPARy and does not bind to any of the isolated domains (N-terminal region, the ligand-binding domain or the C-terminus). This result suggests that PRDM16 makes crucial physical contacts in at least two domains of PPARy. PRDM16 binds via its two zinc finger regions to PPARy. Mass spectrometry analysis of PRDM16 transcriptional complexes showed that PRDM16 associates with p300/CBP in brown adipocyteswhether this acetyltransferase complex mediates activation of PPARs remains to be tested. Repression of Myogenesis. PRDM16 potently represses myogenic differentiation when ectopically expressed in C2C12 or primary satellite cell-derived myogenic cells (2). PRDM16 also associates with a known repressor complex containing C-terminal binding protein-1 or -2 (CtBP-1, 2) to suppress white-fat gene expression (4). The requirement for CtBP in the PRDM16-dependent repression of muscle specific genes was examined in C2C12 myoblasts using a mutant form of PRDM16 that no longer associates with CtBPs but retains its capacity to induce brown adipogenic genes (4). Interestingly, the CtBP binding mutant form of PRDM16, PRDMie''^'was unable to repress some muscle specific genes (e.g. myogenin) but retained its repressive effect on others (e.g. MyoD). This result suggests that a PRDM16/CtBP complex mediates the repression of some but not all muscle-related genes. To define other factors that may contribute to the repression of muscle genes by PRDM16, we performed a mass spectrometry analysis of the PRDM16 complex in C2C12 cells. A number of candidate proteins that may be involved in the PRDM16-driven cell fate transition between myoblasts and brown fat cells were identified.

目标1：我们检验了PRDM16可以推动小鼠白脂肪库中功能性棕色脂肪细胞的形成以增加能量消耗并防止肥胖和冷暴露的假设。 饮食引起的肥胖症（DIP）。雄性AP2-PRDM16和同窝控制动物的第一批队列目前是在一项长达16周的研究中，他们正在食用含有高脂肪的饮食（脂肪的55％卡路里）。在过去的一年中，出于必要性，我们将转基因小鼠系列跨入C57BIACK6遗传背景。转基因小鼠是在FVB背景中产生的，这种近交性菌株不容易发生高脂肪饮食诱导的肥胖症。直接在C57Black6菌株中产生转基因线的成功没有任何成功。 C57Black6小鼠是提出代谢实验类型的金标准。 冷曝光。急性和慢性冷暴露实验将在C57BLACK6遗传背景中的单独的转基因和野生型同窝窝室中进行。 表达成纤维细胞的PRDM16的移植。目的是使用表达PRDM16的前体细胞制定自体移植方案，以形成动物中功能性棕色脂肪组织（BAT）的异位沉积物。假设是，这种“合成”蝙蝠将安全有效地筹集能量消耗以减少肥胖症。在新数据中，白脂肪中基质血管（SV）细胞中的PRDM16表达在皮下移植后促进了体内的脂肪形成，但是移植始终很小 （1-2毫米），具有白色脂肪的单眼外观，没有表达热标记UCP1。 此外，表达PRDM16的骨骼肌细胞在后形成了脂肪组织的异位库 皮下移植。该结果表明，prdivi16在致命的肌细胞细胞中的掺杂作用与控制体内脂肪细胞分化的“真实”环境线索兼容。 同时，由Shingo Kajimura领导的机械研究表明，PRDM16结合并强烈共振了C/EBPP（1）。 c/eBP（3在肌原性细胞中表达得很好，在PRDM16驱动的棕色脂肪生成性测定中是遗传所必需的。值得注意的是，来自C/EBPP缺乏的胎儿小鼠的BAT显着显示出热降低的热基因表达和肌肉特异性转录水平的表达，以肌肉特异性转录对P/p/emcle升高到P/p/emcle的水平。幼稚成纤维细胞中PRDM16和C/EBPP的共表达激活了棕色脂肪细胞独有的脂肪生成和热基因程序。此外，这些成纤维细胞在皮下植入后六周内导致小鼠的多眼，UCP1表达和^^ FDG-PET阳性蝙蝠。这些结果表明，包含C/EBPP和PRDM16的转录单元驱动棕色脂肪表型。 AIM 2：我们通过创建和表征组织特异性PRD/WT6缺陷型小鼠来研究BAT和全身代谢中PRDI \/I16的生理需求。 现在，我们已经成功地从prdmid“”“^”^es细胞系（4个独立的细胞克隆）生成了7个高百分比的Agouti嵌合小鼠。这些动物目前正在用野生型小鼠繁殖，以建立目标等位基因的种系传播。 提交赠款提案后，我们意识到Prd/w^6缺陷的老鼠 在波士顿的Brigham＆妇女医院的David R. Beier实验室中产生。敲除动物是围产期致死的，并具有明显的left裂（B. Bjork，D。Beier et a/。，综述）。与Beier Lab合作，我们分析了胎儿PRD/W76缺陷小鼠的BAT表型。如今报道，PF？DM^6缺陷蝙蝠含有大脂肪液滴和热基因表达的显着降低（2）。该结果揭示了组织正常发育中PRDM16的遗传需求。此外，PRDM16^'BAT表达了更高水平的骨骼肌（SL \/L）特异性基因（2）支持SIVL和BAT可能是由共同谱系引起的概念（3）。 尽管PRDA/F76缺陷的蝙蝠表现出基因表达的失调模式，但它保留了明显的棕色脂肪属性，并且仍然可以识别为BAT。因此，体内PRDM16的慢性损失不会导致蝙蝠分化的总损失。这与基于SHRNA的细胞培养敲低研究不同，在这种敲低研究中，prdivlie在原发性棕色成脂蛋白前体中的耗竭阻止了棕色脂肪形成并促进了明显的骨骼肌发生（2）。因此，我们假设含有PR域蛋白质家族的另一个密切相关的成员可以部分弥补BAT发育中PRDM16的慢性损失。实际上，PRDM3和PRDM16通过序列比较密切相关，并且具有显着的序列相似性，尤其是在两个保守的锌指DNA/蛋白结合结构域中。 因此，我们在细胞培养分析中测试了PRDM3是否在棕色脂肪形成中起作用。 初步研究表明，PRDM3是PPARY2和成脂分化的关键调节剂。 PRDM3在非辅助成纤维细胞（例如NIH-3T3细胞）中的异位表达刺激脂肪形成，包括以cAMP依赖性的方式（如UCP1和PGC-1A）诱导某些蝙蝠相关的基因。 AIM 3：我们在以下方面研究了PRDM16功能的机理基础：（1）通过PPARY激活脂肪形成，（2）通过与CTBP1/2结合来抑制肌发生。- PPAR的激活。现在，我们已经报道了Prdivi16是PPARY和PPARA的强大且依赖配体的共激活因子（2）。此外，PPARY的激动作用对于PRDM16在培养的肌细胞中的掺杂作用是必需的。体外结合实验表明，Prdivi16仅能够与全长PPARY结合，并且不与任何分离的域（N末端区域，配体结合域或C端）结合。该结果表明，PRDM16在至少两个PPARY领域中引起了至关重要的身体接触。 PRDM16通过其两个锌指的区域结合与PPARY结合。 PRDM16转录复合物的质谱分析表明，PRDM16与棕色脂肪细胞中的P300/CBP相关，这些乙酰基转移酶复合酶介导了PPAR的激活，尚待测试。 抑制肌发生。当在C2C12或原代卫星细胞衍生的肌源性细胞异位表达时，PRDM16会有效抑制肌源分化（2）。 PRDM16还将含有C末端结合蛋白-1或-2（CTBP-1、2）的已知抑制剂复合物缔合以抑制白色FAT基因表达（4）。 在C2C12成肌细胞中，使用了一种不再与CTBP相关联的突变体形式，在C2C12成肌细胞中检查了CTBP对肌肉特异性基因的抑制作用，但保留其诱导棕色脂肪基因的能力（4）。有趣的是，prdm16的CTBP结合突变体形式，prdmie''^'无法抑制某些肌肉特定基因（例如肌蛋白），但对其他肌肉（例如myod）保留了抑制作用。该结果表明，PRDM16/CTBP复合物介导了某些但不是所有与肌肉相关的基因的抑制。为了定义可能导致PRDM16抑制肌肉基因的其他因素，我们对C2C12细胞中的PRDM16复合物进行了质谱分析。鉴定了许多可能参与肌细胞和棕色脂肪细胞之间PRDM16驱动的细胞命运过渡的候选蛋白。