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: 7925759
• 负责人: Patrick Seale
• 金额: $ 24.9万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7925759
• 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; 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; 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：我们测试了这样的假设：PRDM16 可以驱动小鼠白色脂肪库中功能性棕色脂肪细胞的形成，从而提高能量消耗并防止肥胖和寒冷暴露。 饮食引起的肥胖（DIP）。第一批雄性 aP2-PRDM16 和同窝对照动物目前正处于一项为期 16 周的研究的第 6 周，其中它们正在摄入高脂肪饮食（55% 卡路里来自脂肪）。在过去的一年里，出于必要，我们将转基因小鼠品系与 C57Biack6 遗传背景进行了回交。转基因小鼠是在 FVB 背景下产生的，这种近交品系不易出现高脂肪饮食引起的肥胖。我们没有成功地直接在 C57Black6 菌株中生成转基因品系。 C57Black6 小鼠是代谢实验类型的黄金标准。 冷暴露。急性和慢性冷暴露实验将在具有 C57Black6 遗传背景的转基因和野生型同窝仔鼠的单独群组中进行。 表达 PRDM16 的成纤维细胞的移植。目标是开发一种自体移植方案，使用表达 PRDM16 的前体细胞在动物体内形成功能性棕色脂肪组织 (BAT) 的异位沉积。假设这种“合成”最佳可行技术能够安全有效地增加能量消耗以减少肥胖。在新的数据中，来自白色脂肪的基质血管（SV）细胞中PRDM16的表达促进了皮下移植后体内脂肪的形成，但是移植物总是很小 （1-2毫米），具有白色脂肪的单房外观并且不表达产热标记物UCP1。 此外，表达 PRDM16 的骨骼肌成肌细胞在 皮下移植。该结果表明PRDIVI16在定型成肌细胞中的脂肪形成作用与控制体内脂肪细胞分化的“真实”环境线索相一致。 与此同时，Shingo Kajimura 领导的机制研究表明 PRDM16 结合并强烈共激活 c/EBPp (1)。 c/EBP(3 在生肌前体细胞中良好表达，并且是 PRDM16 驱动的棕色脂肪形成测定所必需的。值得注意的是，来自 c/EBPp 缺陷胎儿小鼠的 BAT 显示生热基因表达显着降低，肌肉特异性转录物水平升高，类似于在 P/?OM) 6 缺陷组织中观察到的情况。 PRDM16 和 c/EBPp 在幼稚成纤维细胞中的共表达激活棕色脂肪细胞特有的脂肪形成和产热基因程序。此外，这些成纤维细胞在皮下植入六周后在小鼠中产生多房、表达UCP1和FDG-PET阳性的BAT。这些结果表明含有 c/EBPp 和 PRDM16 的转录单位驱动棕色脂肪表型。 目标 2：我们通过创建和表征组织特异性 PRD/Wt6 缺陷小鼠，研究 BAT 和全身代谢中 PRDI\/I16 的生理需求。 我们现在已经成功地从 PRDMId"""^"^'* ES 细胞系（4 个独立的细胞克隆）中产生了 7 只高百分比刺鼠嵌合小鼠。这些动物目前正在与野生型小鼠繁殖，以建立目标等位基因的种系传播。 提交资助提案后，我们意识到 PRD/W^6 缺陷小鼠 波士顿布莱根妇女医院的 David R. Beier 实验室生成。基因敲除动物在围产期是致命的，并且具有明显的腭裂（B. Bjork、D. Beier 等人，评论中）。我们与 Beier 实验室合作，分析了 PRD/W76 缺陷小鼠胎儿的 BAT 表型。据目前报道，Pf?DM^6 缺陷的 BAT 含有大的脂滴，并且生热基因的表达显着减少 (2)。这一结果揭示了组织正常发育中 PRDM16 的遗传需求。此外，PRDM16'BAT表达较高水平的骨骼肌(S11/1)特异性基因(2)，支持SIV1和BAT可能源自共同谱系的观点(3)。 尽管 PRDA/f76 缺陷的 BAT 表现出基因表达失调模式，但它保留了显着的棕色脂肪属性，并且仍然可被识别为 BAT。因此，体内PRDM16的慢性丧失不会导致BAT分化的完全丧失。这与基于 shRNA 的细胞培养敲除研究不同，在该研究中，原代棕色脂肪形成前体中 PRDIVlie 的消耗会阻止棕色脂肪形成并促进明显的骨骼肌生成 (2)。因此，我们假设含有 PR 结构域的蛋白质家族的另一个密切相关的成员可能部分补偿 BAT 发育过程中 PRDM16 的慢性丢失。事实上，通过序列比较，PRDM3 和 PRDM16 密切相关，并且具有显着的序列相似性，特别是在两个保守的锌指 DNA/蛋白质结合域内。 因此，我们在细胞培养测定中测试了 PRDM3 是否在棕色脂肪生成中发挥作用。 初步研究表明PRDM3是PPARY2和脂肪形成分化的关键调节因子。 PRDM3 在非成脂成纤维细胞（如 NIH-3T3 细胞）中的异位表达会刺激脂肪生成，包括以 cAMP 依赖性方式诱导一些 BAT 相关基因（如 UCP1 和 PGC-1a）。 目标 3：我们研究 PRDM16 功能的机制基础：(1) 通过 PPARy 激活脂肪生成和 (2) 通过与 CtBP1/2 结合抑制肌肉生成。- PPAR 的激活。我们现已报道，PRDIVI16 是 PPARy 和 PPARa 的强配体依赖性共激活剂 (2)。此外，PPARγ 的激动对于 PRDM16 在培养的成肌细胞中的脂肪形成作用是必要的。体外结合实验表明，PRDIVI16 只能与全长 PPARγ 结合，不能与任何分离的结构域（N 端区域、配体结合结构域或 C 端）结合。这一结果表明 PRDM16 在 PPARy 的至少两个域中进行重要的物理接触。 PRDM16 通过其两个锌指区域与 PPARγ 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，这种乙酰转移酶复合物是否介导PPARs的激活还有待测试。 抑制肌生成。当 PRDM16 在 C2C12 或原代卫星细胞衍生的肌原细胞中异位表达时，可有效抑制肌原分化 (2)。 PRDM16 还与已知的含有 C 末端结合蛋白-1 或 -2 (CtBP-1, 2) 的阻遏蛋白复合物结合，以抑制白色脂肪基因表达 (4)。 使用 PRDM16 的突变形式在 C2C12 成肌细胞中检查了 PRDM16 依赖性肌肉特异性基因抑制中对 CtBP 的需求，该突变形式不再与 CtBP 相关，但保留了诱导棕色脂肪形成基因的能力 (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异性基因（例如肌细胞生成素），但保留了对其他基因（例如 MyoD）的抑制作用。这一结果表明 PRDM16/CtBP 复合物介导一些但不是全部肌肉相关基因的抑制。为了确定可能有助于 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。鉴定出许多可能参与 PRDM16 驱动的成肌细胞和棕色脂肪细胞之间细胞命运转变的候选蛋白。