ABSTRACT: The focus of this research is in the area of biological clocks. Importance: 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. The objective of this work is to understand how circadian rhythms affect adipocyte biology. To delineate the relationship between the cellular circadian system and adipocyte biology in the absence of organismal cues, circadian output was characterized by transcriptionally profiling in vitro differentiated adipocytes from inguinal adipose tissue over 3 circadian days with a 2-hour resolution via RNAseq. Goal: Determine what aspects of adipocyte biology and environmental stimuli can be influenced by time-of-day. Impact: 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. Purpose: The goals of this study are to understand the molecular basis of the biological clock in mammals and the means through which it controls metabolism. PER2::LUC transgenic mice were used as a source of adipocyte stem cells for studies on the role of the clock in adipocyte biology. Methods: There are two biological replicates. 4 male mice aged 8-12 weeks under 12:12 light-dark cycle and standard chow were used per replicate. The inguinal stromal vascular fraction (SVF) was isolated post-mortem at ZT10*lights on, resting phase, 10am*. Isofluorine anesthesia and cervical dislocation were the method of sacrifice. Pre-adipocytes were isolated as in (Oeckl 2020, DOI: 10.1016/ j.xpro.2020.100118) with modifications and seeded onto three 35mM plastic bottom tissue culture plates (MatTek) in "base media" (DMEM F/12 + GlutaMAX (Gibco xxxx-018) with penicillin/streptomycin and 10% fetal bovine serum (Gibco: Ref#10437-028). Cells were passaged every 48 hours for a total of 3 passages with a split ratio of 1:2.8. In final passage, cells were seeded into 35mM plastic dishes (MatTek). After confluence, differentiation was induced with dexamethasone, rosiglitazone and IBMX, biotin, insulin, pantothenate ( "differentiation media"). After 4 days of differentiation, dex, rosi and IBMX were removed from the media ( "maintenance media"). After 4 days in culture, cells were synchronized with 50% FBS, 50% "base media" and 10uM forskolin for 2-2.5hours. Media was switched to "maintenance media" containing 0.01% bright d-luciferin (GoldBio #LUCK-100) in batches of 4 dishes, topped with glass microscopy slides and sealed with vacuum grease before loading into a Lumicycle housed in a 37C, 0%CO2 incubator. Circadian parameters including amplitude were determined using LumiCycle Analysis software (Actimetrics, v.2.44).
