Neuronal control of body weight and size by the Gbeta5-R7 signaling complex

Gbeta5-R7 信号复合物对体重和体型的神经元控制

• 批准号: 10001791
• 负责人: Vladlen Z Slepak
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001791
• 关键词: Ablation; Acute; Adult; Affect; Agonist; Alleles; Animals; Antibodies; Behavior; Beta Cell; Biochemical; Biological Assay; Biophysics; Body Size; Body Weight; Brain; CRISPR/Cas technology; Cardiovascular Diseases; Cardiovascular system; Cell Line; Cell Nucleus; Cells; Comorbidity; Complex; Control Animal; Data; Diabetes Mellitus; Diagnostic; Disease; Energy Intake; Engineering; Etiology; Event; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GNB5 gene; GTP-Binding Protein Regulators; GTP-Binding Protein beta Subunits; GTP-Binding Proteins; Gene Transfer; Genes; Genetic; Goals; Growth; Health; Hormone secretion; Hormones; Human; Human Genetics; Hypothalamic Hormones; Hypothalamic structure; Image Analysis; In Situ; In Vitro; Injections; Insulin; Investigation; Kidney Diseases; Knock-out; Lead; Link; LoxP-flanked allele; Malignant Neoplasms; Measurement; Mediating; Messenger RNA; Metabolic Diseases; Metabolic syndrome; Metabolism; Methods; Microscopic; Molecular; Mus; Muscarinic Acetylcholine Receptor; Mutation; Neuraxis; Neuroendocrinology; Neuroglia; Neurologic; Neurons; Neurosciences; Neurosecretion; Neurosecretory Systems; Neurotransmitters; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organ; Pancreas; Pathologic; Pathway interactions; Pattern; Peripheral; Pharmacology; Phenotype; Phosphorylation; Physiological; Play; Population; Proteins; RGS Proteins; Regulation; Reproduction; Research; Risk Factors; Role; Second Messenger Systems; Serum; Signal Transduction; Signaling Protein; Slice; Stimulus; Stroke; Structure; Structure of beta Cell of islet; System; Technology; Testing; Therapeutic; Tissues; United States; Viral; Viral Vector; Virus; Weight Gain; base; cholinergic; design; dimer; effective therapy; experimental study; hormonal signals; hormone regulation; improved; in vivo; insight; insulin secretion; kinase inhibitor; knock-down; knockout gene; mutant; nervous system disorder; novel; overexpression; promoter; protein complex; protein function; protein protein interaction; real-time images

Abstract Obesity is a major co-morbidity factor in diabetes, cancer, cardiovascular, neurological and other diseases. One group of genes recently linked to obesity in humans are genes encoding the R7 family of regulator of G protein signaling proteins (RGS6, 7, 9 and 11) and Gnb5, which encodes the G protein beta subunit G5. G5 and R7 proteins form obligatory dimers, so that the Gnb5 knockout causes complete degradation of the entire G5-R7 complex. This proposal studies how a deficiency in the Gnb5 and R7 genes can lead to obesity; the ultimate goal is to discover effective treatments of disorders associated with neuroendocrine pathways regulated by these genes. The proposed research plan builds upon earlier discoveries that together constitute a strong premise for this study. Ablation of one Gnb5 allele leads to obesity and metabolic syndrome in mice; this finding was later confirmed by human genetics. Subsequent mechanistic investigations in this lab identified a novel role for the G5-R7 complex in pancreatic beta cells where G5-R7 strongly promoted insulin secretion. Brain is the main organ regulating body weight, and the expression level of G5-R7 is much higher than in the pancreas or any other peripheral tissues. These considerations lead to the hypothesis that G5-RGS7 regulates body weight via its function in the CNS, where as in the insulin-secreting beta cells, G5-R7 can regulate hormone and/or neurotransmitter secretion by neurons. In support of this hypothesis, recent studies in the lab demonstrated that local knockout of Gnb5 in the hypothalamus using LoxP/Cre technology caused a dramatic increase of body weight and adiposity in adult mice. The proposed research will develop this discovery. Specific Aim 1 will analyze the effects of Gnb5 ablation in specific hypothalamic nuclei and types of neurons on body weight and levels of various hormones. Overexpression of Gnb5 using viral-mediated gene transfer will be performed to rescue obesity. Aim 2 will determine which hormones and signaling proteins (i.e., GPCRs) are co-expressed with Gnb5 in hypothalamic neurons. Tissue and acutely cultured neurons will be examined for secretion of specific hormones and signaling. Specific Aim 3 will study molecular events affected by G5-R7 via its knockout (CRISPR/Cas9), knockdown and overexpression in cell lines that endogenously express M3R and G5-R7. Experiments will focus on signaling, structure-function analysis of G5-R7 and signal-stimulated secretion utilizing pharmacological agents and biochemical and biophysical assays. Through unraveling a novel mechanism that regulates neuronal functions, this project may have a strong impact on understanding etiology of obesity and other neuroendocrine disorders.

抽象的 肥胖是糖尿病、癌症、心血管、神经系统和其他疾病的主要共病因素 疾病。最近与人类肥胖相关的一组基因是编码 R7 家族的基因 G 蛋白信号蛋白（RGS6、7、9 和 11）和 Gnb5（编码 G 蛋白 beta）的调节因子 亚基 G5。 G5 和 R7 蛋白形成强制性二聚体，因此 Gnb5 敲除会导致完全 整个 G5-R7 复合物的降解。该提案研究了 Gnb5 和 R7 基因缺陷如何影响 可能导致肥胖；最终目标是发现与以下疾病相关的有效治疗方法 神经内分泌途径受这些基因调节。 拟议的研究计划建立在早期发现的基础上，这些发现共同构成了强有力的前提 对于这项研究。消除一个 Gnb5 等位基因会导致小鼠肥胖和代谢综合征；这一发现是 后来被人类遗传学证实。该实验室随后的机制研究确定了 胰腺β细胞中的G5-R7复合物，其中G5-R7强烈促进胰岛素分泌。大脑是 是调节体重的主要器官，G5-R7的表达水平远高于胰腺或胰腺 任何其他外周组织。这些考虑导致了 G5-RGS7 调节身体的假设 通过其在 CNS 中的功能来调节体重，与分泌胰岛素的 β 细胞一样，G5-R7 可以调节激素 和/或神经元分泌的神经递质。为了支持这一假设，实验室最近的研究 证明使用 LoxP/Cre 技术局部敲除下丘脑中的 Gnb5 会导致显着的 成年小鼠体重和肥胖的增加。拟议的研究将进一步发展这一发现。 具体目标 1 将分析 Gnb5 消融对特定下丘脑核团和神经元类型的影响 体重和各种激素的水平。使用病毒介导的基因转移过度表达 Gnb5 将 来拯救肥胖症。目标 2 将确定哪些激素和信号蛋白（即 GPCR）是 与下丘脑神经元中的 Gnb5 共表达。将检查组织和急性培养的神经元 特定激素和信号的分泌。具体目标 3 将研究受 G5-R7 影响的分子事件 通过在内源性表达 M3R 的细胞系中进行敲除 (CRISPR/Cas9)、敲低和过表达 和G5-R7。实验将侧重于信号传导、G5-R7 的结构功能分析和信号刺激 利用药物制剂和生物化学和生物物理测定进行分泌。通过解开一个 调节神经元功能的新机制，该项目可能会对理解产生重大影响 肥胖和其他神经内分泌疾病的病因学。