Circadian Regulation of In Vitro Differentiated Adipocytes

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.