In this work, we conducted two experiments, and the schedules used in both of which are detrimental to circadian rhythms. However, the underlying mechanisms are different: experiment #1 provided a non-24 h cycling condition, while experiment #2 yielded highly frequent shifts. We demonstrate that these two schedules may cause extensive alterations of blood/serum variables, suggesting that circadian rhythmicity has been severely influenced.
In both experiments, the participants lived in the isolation condition, and most of the environmental cues including lighting, noise, ambient temperature and workload were controllable so that we could focus on the effects of circadian misalignment. Instead, in real condition like submarine, as the microenvironment differs between different locations or posts on board, the effects of circadian misalignment might be hard to be addressed .
The albumin level and total bilirubin level decreased in experiment #1 but increased in experiment #2, while the triglyceride level increased in experiment #1 but decreased in experiment #2 (Fig. 2,3; supplemental Tables 1,2), which may be owing to the individual difference or the different effects of between non-24-h schedule and rotatory schedule. Changes in metal elements (sodium, iron) and molecules associated with metabolism (glucose, cholesterol, urea and creatinine) were also found in one or both experiments although most of the changes are still within the normal ranges. By contrast, in experiment #1, the levels of total bile acids of five subjects were higher than the normal range (0.1–10.0 µmol/L) after the experiment (Fig. 2f; supplemental Table 1), suggesting that risk of dysfunctions in liver or hepatic duct should be taken into consideration .
Inorganic Pi is a vital component of bone mineralization, phospholipids in membranes, nucleotides that provide energy and serve as components of DNA and RNA, and phosphorylated intermediates in cellular signaling . It is also required for skeletal development, mineral metabolism, and diverse cellular. The level of serum Pi is maintained within a narrow range - the normal plasma Pi concentration is 0.81–1.45 mmol/L, which is regulated by multiple factors, e.g., intestinal absorption, exchange with intracellular and bone storage pools, renal tubular reabsorption, and their interactions [28, 32]. Plasma Pi imbalance, including hypophosphatemia and hyperphosphatemia, are associated with disorders in many organs. Occurrence of hyperphosphatemia is reported in renal failure, hemolysis, tumor lysis syndrome, and rhabdomyolysis [33, 34].
LC20A1 and SLC20A2 are members of the mammalian type-III inorganic Pi transporters encoded by the SLC20 genes, which regulate cellular Pi concentration, Pi sensing, and transcription of downstream genes . These two factors are the only members of this family in humans and they are conserved in many kingdoms including fungi, bacteria and plants, animals [35, 36]. In mammals, SLC20A1 and SLC20A2 are highly expressed in brain although they are ubiquitously expressed [27, 37]. Mutations in SLC20A2 are associated with Fahr’s Disease, characterized by the symmetric calcification in the basal ganglia and other brain regions, and this disease contains a wide spectrum of neuropsychiatric symptoms including headaches, psychiatric disorders, and movement disorders [27; 38]. In this study, some DEMs targeting SLC20A1 and SLC20A2 were identified, suggesting that frequently rotatory schedules might lead to Pi imbalance due to circadian misalignment.
miRNAs constitute a post-transcriptional layer in the regulation of circadian clock and sleep homeostasis [16, 39, 40]. In addition to Pi metabolism, we identified a substantial set of functional miRNAs which are implicated in multiple pathways or physiological processes, including circadian clock and sleep homeostasis, suggesting that these miRNAs may contribute to the misaligned circadian rhythms and affected sleep homeostasis. These DEMs may be causative or resultant of the disturbed circadian rhythms. In addition, DEMs associated with cancer, neural function and several metabolic pathways were also identified, which suggests that desynchronized circadian rhythms may increase the risks of many potential disorders through modulating the expression of corresponding miRNAs.