Circulating Exosomal miRNAs Signal Circadian Misalignment to Peripheral Metabolic Tissues

Abstract

Night shift work increases risk of metabolic disorders, particularly obesity and insulin resistance. While the underlying mechanisms are unknown, evidence points to misalignment of peripheral oscillators causing metabolic disturbances. A pathway conveying such misalignment may involve exosome-based intercellular communication. Fourteen volunteers were assigned to a simulated day shift (DS) or night shift (NS) condition. After 3 days on the simulated shift schedule, blood samples were collected during a 24-h constant routine protocol. Exosomes were isolated from the plasma samples from each of the blood draws. Exosomes were added to naïve differentiated adipocytes, and insulin-induced pAkt/Akt expression changes were assessed. ChIP-Seq analyses for BMAL1 protein, mRNA microarrays and exosomal miRNA arrays combined with bioinformatics and functional effects of agomirs and antagomirs targeting miRNAs in NS and DS exosomal cargo were examined. Human adipocytes treated with exosomes from the NS condition showed altered Akt phosphorylation responses to insulin in comparison to those treated with exosomes from the DS condition. BMAL1 ChIP-Seq of exosome-treated adipocytes showed 42,037 binding sites in the DS condition and 5538 sites in the NS condition, with a large proportion of BMAL1 targets including genes encoding for metabolic regulators. A significant and restricted miRNA exosomal signature emerged after exposure to the NS condition. Among the exosomal miRNAs regulated differentially after 3 days of simulated NS versus DS, proof-of-concept validation of circadian misalignment signaling was demonstrated with hsa-mir-3614-5p. Exosomes from the NS condition markedly altered expression of key genes related to circadian rhythm in several cultured cell types, including adipocytes, myocytes, and hepatocytes, along with significant changes in 29 genes and downstream gene network interactions. Our results indicate that a simulated NS schedule leads to changes in exosomal cargo in the circulation. These changes promote reduction of insulin sensitivity of adipocytes in vitro and alter the expression of core clock genes in peripheral tissues. Circulating exosomal miRNAs may play an important role in metabolic dysfunction in NS workers by serving as messengers of circadian misalignment to peripheral tissues.

The Mystery of Red Blood Cells Extracellular Vesicles in Sleep Apnea with Metabolic Dysfunction

“…EVs released from parental cells may interact with target cells, leading to the subsequent influence of target cell behavior and phenotype features [ 109 ]. In plasma, we have isolated EVs from adult, children, and animals exposed to sleep apnea [ 107 , 110 , 111 , 112 , 113 , 114 , 115 ].…”

“…The most common methods for isolation of EVs or exosomes can be applied from one source to another with some modification. The following are the most common methods used for isolating EVs: ultracentrifugation, sequential centrifugation, density gradient, filtration, sucrose gradient precipitation, polyethylene glycol, acetate, size-exclusion column (Sepharose gel, Sephadex affinity-based capture), and magnetic beads [ 110 , 111 , 112 , 113 , 125 , 126 , 127 , 128 ]. Each of these methods has advantages and disadvantages which include time-consuming, labor-intensive, and expensive equipment.…”

“…There are different methodologies to characterize and/or quantify EVs as shown Figure 4 . EVs morphology can be characterized using conventional electron microscopy which provides information on their structure and size as a cup-shaped morphology [ 112 , 113 ]. Quantitative methods to detect EVs include nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) [ 148 , 149 ].…”

“…Among these proteins Alix, Flotillin, and TSG101 can participate in exosome biogenesis rendering exosomes distinct from the ectosomes released upon plasma membrane shedding. The presence of known cellular proteins in exosome preparations from various cellular sources have been analyzed by Western blotting and by fluorescence-activated cell sorting (FACS) analysis of exosome-coated beads [ 107 , 112 , 115 ] based on their components including integrins and tetraspanins (CD63, CD9, and CD82) Figure 4 . The function of these proteins in EVs is unknown at present.…”

The Multifunctionality of Exosomes; from the Garbage Bin of the Cell to a Next Generation Gene and Cellular Therapy

“…Exosome associated miRNAs have been extensively profiled from virtually all possible sources including plasma [12], cerebrospinal fluid [13], milk [14], semen [15], urine [16], amniotic fluid [17] and bronchoalveolar lavage [18]. The miRNA composition in plasma derived exosomes is highly sensitive to change in microenvironment like exposure to gamma rays [19], cigarette smoke [20] and circadian rhythm [21]. Differentiation into specific cell lineages is also influenced by exosome miRNA profiles.…”