The circadian clock is a regulator of many processes in the body including metabolism, immunity and behavior [18,19,20,21,22,23]. The GM contributes to many physiological processes and as recent data show, is no exception to circadian regulation [24,25,26,27].
In this study, we ran our experiments on rats. Due to their sociability, rats are frequently used as a standardized biological test system. They are nocturnal animals, and they adapt to unfavorable environmental conditions and colonize a wide variety of biotopes. Besides this, they remain active all year round and can also adjust their diurnal rhythmicity to the daily routine of the humans with whom they cohabit. According to Aschoff’s rule, diurnal animals kept in constant darkness stay awake for a much longer period of time than they sleep. Conversely, nocturnal animals exposed to constant light spend more time awake than asleep [28].
Rats exposed to constant light showed signs of anxiety and increased horizontal and vertical activities coupled with a strong interest in their environment. They also spent more time awake than asleep and gained approx. 20–30 g in weight per week. Rats housed in darkness were less active and sometimes aggressive. They spent more time asleep than awake and gained weight less efficiently, approx. 10–20 g per week (unpublished data).
The sympathoadrenal system plays a major role in the organism’s response to the changing conditions in the internal and external environments. The proximity of CAs to the sympathetic nervous system has functional significance for maintaining homeostasis in the body. CAs are formed in the brain and other nervous tissues where they act as neurotransmitters. Dopaminergic neurons are incapable of synthesizing NE due to their lack of dopamine-β-hydroxylase. The adrenal glands synthetize and store both E and NE in large quantities in secretory vesicles and are expelled out from the cell via exocytosis. NE is released from all postganglionic sympathetic nerve endings. This multistage process is regulated by the ability of the final products to inhibit the first enzyme involved in the initial stages of synthesis. Thus, DA, NE and E inhibit tyrosine hydroxylase. As these latter products accumulate in the neurons, a state of equilibrium is established when their synthesis and degradation rates become equal.
After 1 month of constant darkness, the amount of DA in the rats’ urine (DD1) decreased almost 2-fold. The level of NE decreased as shown in Fig. 1. As for E, its level in rat urine was no different from that of the control group (LD1). Changes in different light/dark cycles led to changes in the synthesis rate of E precursors and to the stockage of enough NE in vesicles to maintain homeostasis. This was due to night-time NE production, which activated epiphyseal adrenergic receptors, which in turn stimulated the secretion of melatonin accounting for the anti-stress effects. In this case E acts as a neuromodulator.
After 3 months of rats living in desynchronosis, DA, NE and E levels in the constant light group decreased as compared to the control group and the constant lighting group. The disturbance in melatonin synthesis (Constant light inhibits melatonin synthesis) and the development of hypoxia lead to a disruption in the cells energy balance which activated glycogenesis. Consequently, amino acids (Tryptophan, in particular, the precursor of serotonin and serotonin is the precursor of melatonin) are used to synthesize glucose. Since serotonin controls the activity of other neuromediators, alterations in brain serotonin levels causes an adrenal insufficiency. The depletion of adrenalin and its products leads to depression and a decrease in the organism’s adaptive reserves [29].
Constant darkness or constant light, as abnormal lighting conditions, both lead to hypoxia. As evidence for the rats developing hypoxia, we rely on the altered values of lipid peroxidation and on the altered activity of the antioxidant systems of erythrocytes. Lipid peroxidation is a process triggered by reactive oxygen species (ROS) which leads to the generation of lipid peroxides in the cells’ membranes; the lipid peroxidation-induced damage is normally repaired by specialized enzymes. However, the disruption of the established balance between pro- and antioxidants causes much more stable peroxidation products such as MDA to form. The basal MDA level in the body tissues is fairly low, thus any increase in its MDA levels is a potent indicator of lipid peroxidation and of a disbalance in the cells’ redox status. For the above reasons, we measured the MDA levels at 1 and 3 months. Another biomarker of intense lipid peroxidation is the level of CDs which had significantly increased in all the experimental groups by the end of the third month. These changes, which are triggered by hypoxia, are possibly related to changes in the fatty-acid composition of phospholipids of the cells’ membranes of erythrocytes.
Superoxide dismutase (SOD) is a potent enzymatic antioxidant that is considered to be the first line of defense against free radicals superoxide anion. SOD catalyzes the following reaction: O2− + O2− ––––> H2O2 + O2. SOD is found in all oxygen-consuming cells. The rate of reaction catalyzed by SOD is extremely high and is limited only by the superoxide anion’s diffusion rate. SOD deactivates free radicals that may be generated either endogenously as a by-product of the electron transport chain in the mitochondria or exogenously as a result of an interaction with mixed-valent metals, ionized and UV radiation, ultrasound, etc. In our study, SOD activity dropped significantly after 1 month with a decrease of more than 50% in DD1.
The erythrocytes contain another important enzyme involved in antioxidation: selenium-containing GPx. This enzyme catalyzes the reduction of peroxides using a tripeptide called glutathione. G6PD is a key enzyme involved in the pentose phosphate pathway which plays a major role in maintaining the level of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH supplies H+ to glutathione reductase which reduces glutathione disulfide (GSSG) to glutathione (GSH).
Based on the above, we can say that despite a disruption in the redox balance, the activity of the cells’ antioxidant systems does not indicate the presence of oxidative stress, a deteriorative process that causes irreversible destruction of macromolecules. In our case, the ROS serve as a trigger for regulating the bioenergetic processes. Previously, we demonstrated that a change in the dark/light cycle caused an energetic imbalance in the cellular metabolism as a consequence of hypoxia developing [29]. The ROS so generated can be used as activation energy and for modulating enzymatic activity. The ROS may also fulfill the role of an oscillator as a part of a pacemaker in the biochemical and physiological processes in body cells.
Little is known today about the role of hormones in the transcription and posttranslational modification of clock genes, but it has been suggested that clock proteins may be involved in the hormonal regulation of the cells’ metabolic reactions. Thus, the question of the hormones’ potential role in synchronizing circadian oscillators localized in different tissues of the body remains open for discussion. Unlike mammalian peripheral tissues and cultured fibroblasts which both harbor self-sustained molecular clocks, the master circadian clock governing behavioral rhythms is located in the hypothalamic SCN. In these cells, clock genes and their products form transcriptional/translational feedback loops where the proteins BMAL1 and CLOCK through E-box elements transactivate a series of genes, including per and cry. The translated PER and CRY proteins then suppress the function of the BMAL1–CLOCK complex [30, 31]. Posttranslational modifications such as phosphorylation regulate the activity of clock proteins (stability, localization and interaction) [32].
In our study, we established a correlation between Bmal1 concentrations in hypothalamic cells and CA concentrations in rat urine. After 1 month, DA and NE urinary concentrations dropped 2-fold in DD1 which correlates with a 2-fold decrease in Bmal1 concentrations as compared to LD1. After 3 months, we observed a significant decrease in all urinary CA levels in LL3. Bmal1 protein levels in the hypothalamus of the experimental groups were comparable to those in the control group. We suggest the hormonal system, in particular, CAs can be regarded as a synchronization super system of split-level circadian oscillators.
Proteins made from clock genes in most mammalian cells belong to a family of transcription factors containing a Per-Arnt-Sim (PAS) domain which is activated by light, oxygen, and other gases, as well as by steroid and peptide hormones (Additional file 1: Figure S1). Bmal1, the main molecular circadian oscillator, contains this light-sensitive domain. The PAS domain is also present in proteins (Cry, Per, Clock, Hif1α and Hif1β) and is involved in maintaining the cellular metabolic homeostasis. These facts allow us to hypothesize that such proteins participate in the transcription of hormone receptors.
While mRNA levels of BMAL, PER and CRY in the SCN are produced in a rhythmic fashion at specific times of the day, the expression of mRNAs coding for the CLOCK protein occurs constantly.
We also know that for the heterodimer CLOCK/BMAL1 to be functional (when Per and Cry are expressed) the acetylation of chromatin is necessary. Histone acetylation, deacetylation and methylation, which are rhythmically occurring processes, contribute significantly to the regulation of circadian rhythms. CLOCK protein, by participating in the acetylation of the dimer BMAL1, acts by itself as a histone-acetyltransferase.
It is possible that epigenetic changes in the cell’s genome drive the oscillation in BMAL1 and CLOCK proteins as the day is replaced by night.
Only few research papers studied the changes in composition and functional activity of the GM of animals housed in constant light or constant darkness. However, in those studies, the authors did not compare the differences between LL and DD groups and the duration of the experiments did not exceed 4 weeks [33, 34]. In our study, rats housed in both constant light and constant darkness exhibited significant changes in the GM composition. In the experimental groups LL and DD, we observed a decrease in alpha diversity of the GM as compared to the control group LD. There was also a significant change in the taxonomic composition of both LL and DD groups after 1 and 3 months of observation. The most dramatic changes were increases the genera Lactobacillus and Faecalibacterium (LL1/LD1, DD1/LD1, LD3/LD1) and decrease Ruminococcus (for DD1/LD1). After 1 and 3 months, the magnitude of changes in the GM taxonomic composition differed between the experimental groups (LL and DD) and the control group LD, but the direction of these changes was similar. Thus, the changes in the GM of rats, viewed as an adaptation process to the changing lighting conditions, were similarly oriented in both experimental groups. We note that the GM composition of the control group LD differed between the time points 1 and 3 months (LD1/LD3). This is possibly related to an ongoing process of adaptation of animals to the housing conditions.
As for the predicted functional properties of the GM which characterize its metabolic activity, they didn’t change as dramatically as the taxonomic composition. After 1 month, these changes were different between LL1/LD1 and DD1/LD1 groups. The number of significantly altered GM metabolic pathways between the rats housed in constant light (LL1/LD1) was much higher than that of rats housed in darkness (DD1/LD1). The amplitude of these changes however was small and did not exceed 1.2 in most cases. The most pronounced difference was in the phosphotransferase system between LL1/LD1 groups, which increased in LL1 by 1.8. The difference in fructose and mannose metabolism between LL1/LD1 groups was the second highest and it increased by 1.35. This increase in the phosphotransferase and fructose and mannose metabolism could be indicative of increase in overall metabolism of rats and their GM resulting from increased wakefulness. The increase in the metabolism of retinol in the GM of rats in DD1/LD1 is possibly due to a rise in the activity of the visual system of rats in the darkness.
After 3 months of the experiment, the differences in species diversity and functional capacity of the GM between the experimental and control groups were leveled out (Fig. 5). We believe that after 3 months the rats finally started adapting to their new environment.
