Circadian Regulation of In Vitro Differentiated Adipocytes

摘要 代谢失调是导致慢性阻塞性肺疾病的主要可预防的危险因素。 与疾病有关的死亡。更具体地说，慢性肥胖与脂肪细胞肥大相关 和增生，这两者都可能是昼夜调节的。所有的生物钟都是细胞内在的， 而组织特异性的昼夜节律振荡器则通过微调 营养利用;脂肪组织对微环境变化的反应， 方式产热脂肪细胞可以在营养过程中将能量从ATP生产中转移出去 通过破坏电化学质子梯度，在一个被称为非质子的过程中产生热量， 颤抖产热（NST）。产热脂肪细胞有时能够细胞- 自主感测环境温度并采用可逆产热曲线。的 生物钟在这种产热可塑性中的重要性还没有得到很好的理解， 决定采用这种状态。这项工作的目的是了解昼夜节律是如何 影响脂肪细胞生物学，尤其是产热可塑性。来描述 细胞昼夜节律系统和脂肪细胞生物学在缺乏有机体线索的情况下， 输出将在特定目标1中通过体外分化的转录谱来表征。 在3个昼夜中，以2小时的分辨率，通过 RNAseq。通过这种方式，我将确定脂肪细胞生物学和环境刺激的哪些方面 也会受到时间的影响。虽然多细胞性和线粒体丰度不是 作为产热潜能本身的指标，这两种细胞器复杂地参与NST。 为了扩展生热可塑性是时钟控制的假设，我将使用定量的 荧光活细胞显微术表征脂滴和线粒体空间模式 从而描述细胞器形态作为昼夜节律时间的函数。特别目标2 将决定细胞自主时钟在产热中的作用， 产热，使用红外热成像来识别产热的节律（1） 通过与BAM 15解偶联降低生物能量效率的状态，和（2）通过抑制 UCP 1与嘌呤核苷酸。这项提案的长期目标是确定时钟的作用 调节产热作用。这项研究的结果将增加我们对时钟的理解- 控制能量代谢和脂肪细胞功能障碍，推进我们对 体重、能量消耗和慢性病风险之间的非线性关系。

ABSTRACT Metabolic dysregulation is the major preventable risk factor for leading causes of chronic disease-related deaths. More specifically, chronic obesity is correlated with adipocyte hypertrophy and hyperplasia, both of which may be circadianly regulated. All circadian clocks are cell-intrinsic, and circadian oscillators that are tissue-specific control metabolic homeostasis by fine-tuning nutrient utilization; adipose tissue responds to microenvironmental changes in a clock-dependent manner. Thermogenic adipocytes can redirect energy away from ATP production during nutrient excess by disrupting the electrochemical proton gradient, producing heat in a process called Non- Shivering Thermogenesis (NST). Thermogenic adipocytes are sometimes capable of cell- autonomously sensing ambient temperature and adopting a reversible thermogenic profile. The circadian clock's importance in this thermogenic plasticity is not well understood, nor the cellular decision to adopt this state. The objective of this work is to understand how circadian rhythms affect adipocyte biology, especially thermogenic plasticity. To delineate the relationship between the cellular circadian system and adipocyte biology in the absence of organismal cues, circadian output will be characterized in Specific Aim 1 by transcriptionally profiling in vitro differentiated adipocytes from inguinal adipose tissue over 3 circadian days with a 2-hour resolution via RNAseq. In this way I will determine what aspects of adipocyte biology and environmental stimuli can be influenced by time-of-day. Though multilocularity and mitochondrial abundance are not indicators of thermogenic potential per se, these two organelles are intricately involved in NST. To extend the hypothesis that thermogenic plasticity is clock-controlled, I will use quantitative fluorescence live cell microscopy to characterize lipid droplet and mitochondrial spatial patterning and thereby describe organelle morphology as a function of circadian time. In Specific Aim 2 I will determine the cell-autonomous clock’s role in heat production, the quintessential component of thermogenesis, using infrared thermal imaging to identify rhythms in heat production (1) during a state of decreased bioenergetic efficiency via uncoupling with BAM15 and (2) by suppressing UCP1 with purine nucleotides. The long-term goal of this proposal is to determine the clock’s role in regulating thermogenesis. Findings from this study will increase our understanding of clock- controlled energy metabolism and adipocyte dysfunction, advancing our understanding of the non-linear association between weight, energy expenditure and risk in chronic disease.