Dissecting PKA activation of mTORC1 and its function in adipose tissue

剖析 mTORC1 的 PKA 激活及其在脂肪组织中的功能

基本信息

批准号：
10091138
负责人：
SHEILA COLLINS
金额：
$ 6.52万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-22 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10091138
关键词：
Adipocytes Adipose tissue Alanine Amino Acids Antibodies Area Aspartate Behavioral Biochemical Biogenesis Biological Body fat Body mass index Brown Fat CRISPR/Cas technology Calories Catecholamines Cell Culture Techniques Cell model Cells Chronic Disease Clinical Clothing Complex Country Cyclic AMP Cyclic AMP Receptor Protein Cyclic AMP-Dependent Protein Kinases Data Data Set Desire for food Development Disease Endocrine System Diseases Energy Metabolism Engineering Epidemic Event FDA approved FRAP1 gene Fatty Acids Fatty acid glycerol esters Follow-Up Studies Generations Genes Genetic Genetic Transcription Glucose Goals Growth Factor Human In Vitro Insulin Insulin Resistance Intervention Investigation Knock-in Knowledge Laboratory Animals Life Style Lipolysis Longevity Mechanics Metabolic Metabolic Diseases Metabolic syndrome Metabolism Mitochondria Mitogen-Activated Protein Kinases Modeling Molecular Mus Mutation Non-Insulin-Dependent Diabetes Mellitus Obesity Overweight PPAR gamma Pathway interactions Peripheral Phosphoproteins Phosphorylation Phosphorylation Site Phosphotransferases Physiological Population Process Production Proteins Proteomics Protons Public Health Raptors Receptor Activation Regulation Resistance Respiration Ribosomal Protein S6 Kinase Risk Rodent Role Route Safety Scanning Signal Pathway Signal Transduction Sirolimus Site Source Stable Isotope Labeling Sympathetic Nervous System Techniques Technology Testing Therapeutic Thermogenesis Tissues Weight Gain Work absorption beta-adrenergic receptor cell type comorbidity comparative cost experimental study fat burning fighting gain of function gain of function mutation health economics improved in vivo inhibitor/antagonist insight insulin sensitivity kinase inhibitor loss of function mutation novel novel strategies obesity treatment p38 Mitogen Activated Protein Kinase phosphoproteomics prevent programs reconstitution transdifferentiation uncoupling protein 1

项目摘要

Project Summary: Obesity is at epidemic proportions in the US. Over 60% of the population is either overweight (Body Mass Index [BMI] ≥25 to <30 kg/m2) or obese (BMI ≥30 kg/m2), placing them at risk for a large number of chronic diseases, including insulin resistance, metabolic syndrome, and type 2 diabetes. The annual costs of obesity exceed $100 billion, making it one of the most significant public health and economic issues facing the country. Unfortunately, the treatment of obesity is unsatisfactory. Lifestyle and behavioral approaches have a modest, and often transient, effect while FDA-approved therapeutic options targeting appetite or fat absorption have poor tolerability and, in some cases, safety concerns. Thus, there is a critical need for novel approaches to treat obesity. Agents acting via peripheral mechanisms to increase energy expenditure would be valuable. The sympathetic nervous system (SNS) is well-known as an activator of brown adipose tissue (BAT) and the “browning” of cells in white adipose tissue (WAT) depots to increase uncoupled mitochondrial respiration and energy expenditure. Our earlier work established a signaling cascade from β-adrenergic receptors (βARs)  cAMP  protein kinase A (PKA)  p38 MAP kinase (MAPK) to drive the transcription of brown adipocyte genes such as uncoupling protein-1 (UCP1), PPAR-gamma coativator-1α (PGC-1α), and the broader program of mitochondrial biogenesis. We have discovered that the mTOR complex-1 (mTORC1) components mTOR and Raptor are phosphorylated by PKA. This is a highly novel observation, since the `canonical' pathway to mTORC1 is through growth factors and insulin. From in vivo studies in mice we find that blockade of mTORC1 with rapamycin, or genetic deletion of Raptor specifically in adipose tissue, suppresses the ability of the βAR pathway in increase the amount of UCP1-expressing `beige' adipocytes within white fat depots, and dampens UCP1 expression and respiration in interscapular brown fat. The ability of laboratory animals and humans to expand these `beige' adipocytes is closely correlated with resistance to weight gain and improved insulin sensitivity. We have identified the phosphorylation sites on mTOR and Raptor and propose to test the physiological consequences of cells and mice in which Ser791 of Raptor is changed to either Alanine (loss-of-function mutation) or Aspartate (gain-of-function) by engineering a `knock-in' genetic construct. Since we now show that there are two distinct routes to activation of mTORC1, we are also taking an unbiased proteomic approach to identify the unique phosphorylation substrates of mTOR resulting from PKA activation vs insulin. Altogether, these experiments will shed important mechanistic and physiological insight into the steps needed for `beige' cell expansion, as well as the broader ability of the PKA mTORC1 pathway to function in other cell types.

项目概要：肥胖在美国已成为一种流行病，超过 60% 的人口体重超重（体重）。指数[BMI]≥25至<30kg/m2）或肥胖（BMI≥30kg/m2），使他们面临大量慢性病的风险疾病，包括胰岛素抵抗、代谢综合征和 2 型糖尿病肥胖的年度成本。超过1000亿美元，使其成为该国面临的最重大的公共卫生和经济问题之一。不幸的是，生活方式和行为方法对肥胖的治疗并不令人满意。效果通常是短暂的，而 FDA 批准的针对食欲或脂肪吸收的治疗方案却具有耐受性差，在某些情况下还存在安全问题，因此迫切需要新的方法来治疗。通过外周机制作用来增加能量消耗的药物将是有价值的。交感神经系统 (SNS) 是众所周知的棕色脂肪组织 (BAT) 激活剂白色脂肪组织（WAT）库中的细胞“褐变”，以增加解偶联的线粒体呼吸和我们早期的工作建立了 β-肾上腺素受体 (βAR) 的信号级联  cAMP → 蛋白激酶 A (PKA) → p38 MAP 激酶 (MAPK) 驱动棕色脂肪细胞的转录解偶联蛋白-1 (UCP1)、PPAR-gamma cotivator-1α (PGC-1α) 等基因以及更广泛的程序线粒体生物发生。我们发现 mTOR 复合物 1 (mTORC1) 成分 mTOR 和 Raptor 被磷酸化这是一个非常新颖的观察，因为 mTORC1 的“典型”途径是通过生长。通过对小鼠的体内研究，我们发现雷帕霉素或遗传物质可以阻断 mTORC1。特别是在脂肪组织中删除 Raptor，会抑制 βAR 途径增加白色脂肪库中表达 UCP1 的“米色”脂肪细胞数量，并抑制 UCP1 表达和实验动物和人类肩胛间棕色脂肪的呼吸作用扩大了这些“米色”。脂肪细胞与体重增加的抵抗力和胰岛素敏感性的改善密切相关。我们已经确定了 mTOR 和 Raptor 上的磷酸化位点，并建议测试其生理功能 Raptor Ser791 更改为丙氨酸（功能丧失）的细胞和小鼠的后果突变）或天冬氨酸（功能获得）通过设计“敲入”基因结构来实现。 mTORC1 的激活有两种不同的途径，我们还采用公正的蛋白质组学方法来鉴定出 PKA 激活与胰岛素产生的 mTOR 独特磷酸化底物，这些实验将为“米色”所需的步骤提供重要的机械和生理学见解细胞扩增，以及 PKA mTORC1 通路在其他细胞类型中发挥作用的更广泛能力。