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

• 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
• 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

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.

项目摘要： 肥胖症在美国处于流行比例。超过60％的人口要么超重（体重 指数[BMI]≥25至<30 kg/m2）或肥胖（BMI≥30kg/m2），使它们面临大量慢性的风险 疾病，包括胰岛素抵抗，代谢综合征和2型糖尿病。肥胖的年费 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，肥胖的治疗是不令人满意的。生活方式和行为方法有一个谦虚的 而且通常是短暂的，效果是针对靶向食欲或脂肪滥用的FDA批准的治疗选择 耐受性差，在某些情况下也是安全问题。那是对新颖方法的迫切需求 治疗肥胖。通过外围机制作用以增加能量消耗的代理人将是有价值的。这 交感神经系统（SNS）众所周知是棕色脂肪组织（BAT）和 白色脂肪组织（WAT）沉积物中细胞的“褐变”，以增加线粒体呼吸和 能源支出。我们较早的工作从β-肾上腺素能受体（βAR）建立了一个信号级联。 营地蛋白激酶A（PKA）p38地图激酶（MAPK）驱动棕色脂肪细胞的转录 诸如解偶联蛋白-1（UCP1），PPAR-GAMMA COATIVATOR-1α（PGC-1α）和更广泛程序之类的基因 线粒体生物发生。 我们已经发现MTOR复合物-1（MTORC1）组件MTOR和RAPTOR是磷酸化的 由PKA。这是一个高度新颖的观察，因为通往MTORC1的“规范”途径是通过增长 因素和胰岛素。从小鼠的体内研究中，我们发现用雷帕霉素或遗传的MTORC1封锁MTORC1 在脂肪组织中特别删除猛禽，抑制βAR途径增加的能力 白色脂肪沉积物中表达UCP1的“米色”脂肪细胞的数量，该死的UCP1表达和 刺激性棕色脂肪中的呼吸。实验动物和人类扩大这些“米色”的能力 脂肪细胞与对体重增加和改善胰岛素敏感性的抗性密切相关。 我们已经确定了MTOR和RAPTOR上的Photophorylation网站，并提议测试生理 细胞和小鼠的后果将猛禽的Ser791更改为丙氨酸（功能丧失） 突变）或天冬氨酸（功能获得）通过工程“敲入”遗传构建体。因为我们现在表明 MTORC1激活有两种不同的路线，我们还采用公正的蛋白质组学方法 确定由PKA激活与胰岛素引起的MTOR的独特磷酸化底物。共， 这些实验将对“米色”所需的步骤提供重要的机械和物理洞察力 细胞膨胀以及PKAMTORC1途径在其他细胞类型中起作用的更广泛能力。