Metabolic regulators in the mechanisms of caloric restriction

描述(申请人提供)：卡路里限制(CR)通过一种未知的机制在不同的实验生物体中延缓衰老的开始并延长寿命。在小鼠中，CR诱导白色脂肪组织(WAT)的广泛重塑和重新编程，这可能在CR降低全身炎症张力的能力中起重要作用。CR诱导代谢和功能不同的WAT的一个主要事件可能涉及一条新的线粒体适应途径。我们假设参与线粒体适应的代谢调节剂通过改变PGC-11的加工和功能而诱导WAT的重新编程以响应CR。为了测试这一点，我们将使用多方面的方法来研究PGC-11在分子、细胞和系统水平上的调控。有三个具体目的：具体目标1：确定依赖GSK32的PGC-11翻译后修饰的机制和功能后果。CR在WAT中的影响与GSK32活性降低增强PGC-11稳定性的模型一致。为了研究这一点的分子基础，我们将使用NIH-3T3细胞来确定：i)结构性活性或激酶非活性GSK32对PGC-11定位、稳定性和活性的影响，ii)由GSK32磷酸化的PGC-11残基的同一性和完整性要求，以及iii)PGC11破坏框和蛋白降解加工共识序列在指导PGC-11周转和活性方面的意义。特定目的2：确定PGC-11和线粒体适应性在脂肪细胞形态和功能中的作用。Wat中受CR调控的因子也影响3T3-L1前脂肪细胞的成脂，可能是通过影响PGC-11。为了研究这些细胞途径，我们将确定：i)PGC-11在成脂过程中的参与和时间特异性要求，ii)改变PGC-11和GSK32对细胞形态和成脂呼吸转变的影响，以及iii)PGC-11的RNA结合基序在调节丙酮酸激酶促呼吸剪接变体中的作用。具体目的3：确定线粒体适应相关因素对小鼠WAT和血清因子的影响。我们已经提出，CR诱导的GSK32和SIRT1的变化通过PGC-11作用，实现在CR小鼠的WAT中观察到的代谢重编程。我们将确定GSK32抑制(碳酸锂)、SIRT1激活(白藜芦醇)或PGC-11激活(苯扎贝特)在多大程度上能够模拟CR对i)Wat基因表达和交替剪接谱、ii)血清脂质信号分子水平以及iii)血清脂联素、瘦素、抵抗素和内脂素水平的影响。这些拟议的研究有望为PGC-11的调节提供新的机制细节，并对CR重塑WAT提供关键的见解。我们预计，这项研究的意义将扩展到对因放松水务管制而促进的疾病的调查。 公共卫生相关性：卡路里限制(CR)代表了最有力的饮食干预措施，可以延缓包括哺乳动物在内的多种生物的衰老过程。这项研究调查了CR在白色脂肪组织中诱导的影响整体代谢和全身炎症的分子和细胞事件。从这项研究中获得的新的机械论见解与公共卫生面临的两个主要问题有关：与年龄相关疾病的个人人数不断增加，以及与肥胖相关的代谢紊乱的流行。

DESCRIPTION (provided by applicant): Caloric restriction (CR) delays the onset of aging and extends lifespan in diverse experimental organisms through an unknown mechanism. In mice, CR induces an extensive remodeling and reprogramming of white adipose tissue (WAT) that may be important in CR's ability to reduce systemic inflammatory tone. A primary event in CR's induction of a metabolically and functionally distinct WAT may involve a novel pathway for mitochondrial adaptation. We hypothesize that metabolic regulators involved in mitochondrial adaptation induce a reprogramming of WAT in response to CR through alterations in PGC-11 processing and function. To test this we will employ a multifaceted approach to investigate the regulation of PGC-11 at molecular, cellular and systemic levels. There are three specific aims: Specific Aim 1: To determine the mechanism and functional consequence of GSK32 dependent post-translational modification of PGC-11. The impact of CR in WAT is consistent with a model where reduced GSK32 activity enhances PGC-11 stability. To investigate the molecular basis of this, we will use NIH- 3T3 cells to determine: i) the impact of constitutively active or kinase inactive GSK32 on PGC-11 localization, stability and activity, ii) the identity and requirement for integrity of PGC-11 residues phosphorylated by GSK32, and iii) the significance of the PGC11 destruction box and proteolytic processing consensus sequences in directing PGC-11 turnover and activities. Specific Aim 2: To determine the role of PGC-11 and mitochondrial adaptation in adipocyte morphology and function. Factors regulated by CR in WAT also influence adipogenesis of 3T3-L1 preadipocytes, possibly through their impact on PGC-11. To investigate these cellular pathways we will determine: i) the involvement and timing-specific requirement for PGC-11 in adipogenesis, ii) the impact of altered PGC-11 and GSK32 on cell morphology and the adipogenic respiratory transition, and iii) the role of the RNA binding motif of PGC-11 in regulating the pro-respiration splice variant of pyruvate kinase. Specific Aim 3: To determine the impact of factors involved in mitochondrial adaptation on WAT and serum factors in mice. We have proposed that CR-induced alterations in GSK32 and SIRT1 act through PGC-11 to implement the metabolic reprogramming observed in WAT from CR mice. We will determine the extent to which GSK32 inhibition (lithium carbonate), SIRT1 activation (resveratrol) or PGC-11 activation (bezafibrate) can mimic the effect of CR on i) WAT gene expression and alternate splicing profiles, ii) serum levels of lipid signaling molecules, and iii) serum levels of adiponectin, leptin, resistin and visfatin. The proposed studies are expected to provide novel mechanistic details of the regulation of PGC-11 and critical insights into WAT remodeling by CR. We expect that the significance of this study will extend to the investigation of diseases that are promoted by deregulation of WAT. PUBLIC HEALTH RELEVANCE: Caloric restriction (CR) represents the most robust dietary intervention to delay the aging process in multiple organisms including mammals. This study investigates the molecular and cellular events induced by CR in white adipose tissue that influence overall metabolism and systemic inflammation. The novel mechanistic insights to be gained from this study are relevant to two major issues facing public health: the increasing population of individuals with age-associated diseases and the prevalence of metabolic disorders associated with obesity.