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A5934 - Circulating Plasma Exosomes May Signal Circadian Clock Misalignment to Peripheral Tissues
Author Block: A. Khalyfa, Hans P.A. Van Dongen, Shobhan Gaddameedhi, Devon A. Grant, Zhuanhong Qiao, Elena Skornyakov, David Gozal; Pediatrics, University of Chicago, Chicago, IL, United States.
Rationale: A large body of epidemiologic evidence supports an association between night shift (NS) work and metabolic disorders, particularly obesity, insulin resistance, and type 2 diabetes mellitus. The mechanisms underlying NS-induced altered insulin sensitivity are unknown. One possible pathway conveying circadian clock misalignment in NS workers may involve exosome-based intercellular communication. Exosomes are ubiquitous extracellular vesicles that are secreted and transfer bioactive cargos to target cells. We examined changes in miRNA exosomal cargo and the effect of plasma exosomes on insulin sensitivity in differentiated human adipocytes in vitro in healthy young subjects exposed to a simulated NS paradigm. Methods: Fourteen healthy volunteers (4 women, 10 men; age 25.8±3.2 y) participated in a highly controlled laboratory study. Subjects were randomized to either a simulated day shift (DS) or a simulated NS condition (n=7 subjects each). After 3 days on the simulated DS or NS schedule, blood samples were collected during a constant routine protocol, and plasma exosomes were isolated. Exosomes were added to differentiated adipocytes, and insulin-induced pAkt/Akt expression changes were assessed by western blots. Multiple high-throughput approaches were used, including ChIP-Seq analyses for BMAL1, DNA microarrays and exosomal miRNAs arrays combined with bioinformatics. We also assessed the functional effects of agomirs and antagomirs targeting differentially expressed miRNAs in NS and DS exosomal cargo. Results: Human adipocytes treated with NS exosomes showed markedly shifted Akt phosphorylation responses to insulin when compared to DS exosomes, along with overall reduced insulin sensitivity, suggesting that plasma exosomes could be involved in fostering insulin resistance among NS workers. ChIP-Seq of NS and DS exosome-treated adipocytes for BMAL1 showed 42,037 binding sites in DS and 5,538 binding sites in NS, with a significant proportion of BMAL1 targets including genes encoding for central regulators of metabolic processes. A significant and restricted miRNA exosomal signature emerged after NS exposures. NS exosomes markedly altered expression of key genes related to circadian rhythm, such as ARNTL (BMAL1), CRY1, RORA, PER1, PER2, PPARA, and CLOCK in several cultured cell types, including adipocytes, myocytes, and hepatocytes, along with significant changes in 29 genes and downstream gene network interactions. Conclusions: NS schedules induce release of exosomes to the circulation that promote reduction of insulin sensitivity of adipocytes in vitro, and alter the expression of core clock genes in peripheral tissues. Thus, circulating exosomal miRNAs may play important roles in metabolic dysfunction in NS workers by serving as circadian clock messengers to peripheral tissues.