Metabolic regulators in the mechanisms of caloric restriction

描述（由申请人提供）：在多种实验生物中，热量限制（CR）通过一种未知的机制延缓衰老的发生并延长寿命。在小鼠中，CR诱导白色脂肪组织（WAT）的广泛重塑和重编程，这可能对CR降低全身炎症张力的能力很重要。CR诱导代谢和功能上不同的WAT的主要事件可能涉及线粒体适应的新途径。我们假设，参与线粒体适应的代谢调节因子通过改变PGC-11的加工和功能，诱导WAT重编程以响应CR。为了验证这一点，我们将采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。具体目的1：确定GSK32依赖性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。为了研究其分子基础，我们将使用NIH- 3T3细胞来确定：i)组成活性或激酶失活的GSK32对PGC-11定位，稳定性和活性的影响，ii) GSK32磷酸化的PGC-11残基的身份和完整性要求，以及iii) PGC11破坏盒和蛋白水解加工共识序列在指导PGC-11周转和活性中的意义。特异性目的2：确定PGC-11和线粒体适应在脂肪细胞形态和功能中的作用。WAT中受CR调控的因子也可能通过影响PGC-11影响3T3-L1前脂肪细胞的脂肪形成。为了研究这些细胞通路，我们将确定：i) PGC-11在脂肪形成中的参与和时间特异性要求，ii) PGC-11和GSK32改变对细胞形态和脂肪生成呼吸转变的影响，以及iii) PGC-11的RNA结合基序在调节丙酮酸激酶的促呼吸剪接变体中的作用。特异性目的3：确定线粒体适应相关因子对小鼠WAT和血清因子的影响。我们提出CR诱导的GSK32和SIRT1的改变通过PGC-11起作用，实现了CR小鼠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（贝扎布酸）在多大程度上可以模拟CR对以下方面的影响：1)WAT基因表达和交替剪接谱；2)脂质信号分子的血清水平；3)脂联素、瘦素、抵抗素和visfatin的血清水平。这些研究有望为PGC-11调控提供新的机制细节，并为CR对WAT的重塑提供重要见解。我们希望这项研究的意义将扩展到对WAT调控放松促进的疾病的研究。

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.