The Anti-Elixir Triad: Non-Synced Circadian Rhythm, Gut Dysbiosis, and Telomeric Damage

Aging is an inevitable biological process characterized by the gradual decline in physiological functions and the accumulation of cellular damage over time. This phenomenon is influenced by various intrinsic and extrinsic factors, leading to increased susceptibility to age-related diseases such as cardiovascular disorders, neurodegeneration, and metabolic syndromes. Recent research has highlighted the critical roles of circadian rhythms, gut microbiota, and telomere dynamics in aging process. Physiology and behavior in mammals are well-controlled by internal molecular oscillators, a timekeeping system. Thus, an endogenous system regulates sleep-wake phases, heartbeat frequency, blood pressure, core temperature, kidney functions, liver metabolism, and secretion of several hormones [1]. Every organism experiences a free-running period, which refers to the duration it takes for the organism to complete its daily activities and then restart the same cycle without any external environmental cues. This free-running period is a distinctive feature of the circadian clock and varies among different animals [2, 3]. Zeitgebers are external cues that influence the body’s circadian rhythm including physical activity, social interactions, alarm clocks, light-dark cycles, temperature, humidity, tides, and food availability [4, 5]. The process by which the internal circadian clock aligns with these external stimuli (zeitgeber) is called entrainment. Circadian entrainment is a clear adaptation that allows organisms to synchronize their biological rhythms with their environment [6, 7]. Similarly, the gut microbiota, a complex community of microorganisms residing in the gastrointestinal tract, plays a role in metabolic health and immune function, with its composition and diversity often altered during aging [8]. Telomeres, the protective caps at the ends of chromosomes, are crucial for cellular replication and stability; their attrition is a hallmark of aging and cellular senescence [9]. Understanding the interplay among these three components; circadian rhythms, gut microbiota, and telomeres provides valuable insights into the mechanisms underlying aging and offers potential therapeutic avenues for promoting healthy aging.

The period (per) gene was discovered as the first circadian gene through the analysis of Drosophila mutants with aberrant behavioral cycles [10]. The PER protein represses its own gene expression through a feedback mechanism, where the accumulation of PER protein in the nucleus inhibits the transcription of the per gene, thereby regulating its own levels and maintaining the circadian rhythm [11]. Continuous investigations in the mice, have revealed anomalous behavioral patterns in forward genetic screening; a method that involves inducing random mutations and selecting for those that result in specific phenotypic changes. This led to the discovery of the core clock gene [12] which forms a heterodimer with Bmal1 that controls circadian gene expression [13]. Genes and proteins involved in the circadian rhythm architecture are explained in Table 1.

The BMAL1 and CLOCK heterodimer bind to the E-box in the promoters of circadian genes, promoting the transcription of the period genes (PER1, PER2, PER3) and the cryptochrome genes (-CRY1, CRY2) (as shown in Fig. 1). Once transcribed, the PER and CRY proteins heterodimerize, forming a complex that translocate from the cytoplasm into the nucleus. In the nucleus, this complex represses its own transcription by interacting with the CLOCK-BMAL1 heterodimer [14]. The CLOCK-BMAL1 heterodimer further regulates the expression of DBP (D-box binding protein) and NFIL3 genes. The translated DBP and NFIL3 proteins form a heterodimer that binds to the D-box of the RORα/β gene promoter, thereby regulating its expression [15].

The regulation of the PER2 protein involves phosphorylation by casein kinase 1 (CK1), specifically the isoforms CK1ε and CK1δ. This phosphorylation leads to the degradation of PER2 through a process known as the “phosphoswitch,” which relieves repression and allows the CLOCK-BMAL1 heterodimers to initiate the next transcription cycle [16, 17]. CRY2 is phosphorylated at Ser557 by DYRK1A, which subsequently allows GSK-3β to phosphorylate the adjacent Ser553. This dual phosphorylation facilitates the proteasomal degradation of CRY2 [18].

The ubiquitin ligase complex SCFβTrCP1/2 binds to PER2, promoting its proteasomal degradation. Similarly, the SCFFBXL3 complex facilitates the ubiquitination of CRY1 and CRY2. Notably, the association of CRY proteins with FBXL3 leads to their proteasomal breakdown without requiring phosphorylation at Ser553 and Ser557 [19].

The CLOCK-BMAL1 heterodimers also transcribe the genes for nuclear receptors REV-ERBα and REV-ERBβ. These receptors compete with ROR proteins for binding to the ROR element in the BMAL1 gene promoter. While ROR proteins positively regulate BMAL1 expression, REV-ERB proteins exert a negative regulatory effect [20].

The suprachiasmatic nucleus (SCN) located in the anterior hypothalamus serves as the central clock that regulates both physiological and psychological cycles. In contrast, the peripheral clock is an endogenous system responsible for local rhythmic events and driving the 24-h cycle of gene transcription within their specific environments [19, 21]. Consequently, temporal adaptation is crucial for species that need to synchronize their physiology and behavior with relevant environmental cues. The circadian rhythm consists of two key components: the central and the peripheral clock. The central clock, situated in the hypothalamus, synchronizes the internal clock through pathways originating from the retinal hypothalamic tract [22].

The retina functions as a sensory receptor membrane composed of specialized photoreceptors, including rods, cones, and melanopsin, which express intrinsically photosensitive retinal ganglion cells (ipRGCs). While rods and cones are primarily responsible for visual image formation, ipRGCs play a critical role in circadian photo-entrainment [23]. The retino-hypothalamic tract conveys information about external light cues to the SCN [24, 25]. Retino-hypothalamic tract nerve terminals connect to the ventral bilateral SCN from a unique subset of ipRGCs that are widely distributed throughout the retina [26, 27]. The ipRGCs receive photo signals through rods and cones, and their dendritic structure allows them to respond to a broad spectrum of light, triggering a cascade of biological events. The primary pathway involves the transmission of signals from the eye to the brain (Fig. 2), where photon reception is converted into electrical signals that convey information to higher brain regions as action potentials [28, 29].

In natural environments, the most significant synchronizers of circadian rhythms are regular light and temperature cycles. The thermo-phase, characterized by increased temperature, coincides with the light phase, while the chrysoprase phase, marked by decreased temperature, aligns with the dark phase. Thus, dawn is associated with temperature shifts from cold to warm, and dusk corresponds to shifts from warm to cold [30]. Research has demonstrated that the circadian clock can adjust to temperature changes in various species, including plants and animals such as Drosophila, Zebrafish, and Neurospora, which are commonly used as model systems [31‒33]. Some studies suggest that temperature can entrain poikilotherms but not homeotherms, while other research involving chicken pineal cell cultures indicates that temperature can alter the phase of intrinsic rhythmicity in homeotherms [34‒36]. Although the 24-h temperature cycle can generally influence circadian rhythms, its impact on the circadian clock is not as pronounced as that of light [37].

Food anticipatory activity (FAA) rhythms are similar to light-entrainable rhythms; however, the SCN is exclusively responsible for the light-entrainable rhythms, not food anticipatory behavioral rhythms [38, 39]. The FAA that is not related to the SCN is referred to as the food-entrainable oscillator (FEO). However, the anatomical position of the FEO is unspecified, and there is no evidence that the circadian system has a distinct FEO. The feeding-fasting cycle synchronizes peripheral organ circadian rhythms; however, the signal that resets peripheral oscillators in the appropriate organs is not well understood [40].

Under hypocaloric meals, the molecular machinery of the SCN and peripheral organs becomes phase advanced. Nocturnal animals have become partially diurnal as a result of changes in food cues [41]. A study in zebrafish exhibited FAA during temporal food restriction under both light/dark and constant light conditions. Periodic meals fed under distinct light conditions entrained the peripheral liver clock, and the SCN clock appears to be synchronized with the liver exclusively during the light-dark cycle [42]. This study shows that food zeitgebers cannot regulate the circadian clock. Contrarily, glucose infusions in the veins modify clock gene expression in the SCN. Similarly, study subjects in the continuous absence of the photic synchronizer, when given a scheduled meal, experienced a SCN reset [43, 44].

In an intervention study of food zeitgebers, it was discovered that there is a change in the salivary and intestinal microbiota in subjects provided with early or late lunch. Though differences in meal timings and composition lead to the Dim Light Melatonin Onset phase shift and disturbances in the leptin rhythm, the insulin rhythm and plasma triglyceride profiles did not change much [45]. When nocturnally active mice and rodents are solely fed during the day, the gene expression phase completely inverts within a week, and the signals linked with feeding time do not affect the SCN oscillator [46].

Cellular senescence was originally characterized as a genetic standpoint, encompassing an element of the chromosome termed the telomere [47]. They put forward the theory of aging, according to which a human somatic cell can only go through 50 to 70 divisions, after which the cell enters a state of senescence [1]. Hayflick limit or replicative senescence is the term used to define the termination of the cell cycle once the telomere becomes short [48]. DNA ends are secured by telomeres, which enact as a cap to make sure the transfer of genetic information is uncompromised through each replication cycle [49].

The human telomere is composed of tandem repeats, TTAGGG, also called as hexanucleotide sequence and each cell division results in to loss of 50–100 base pairs of the telomere sequence generally [50]. Since DNA polymerase only works in the 5′-3′ direction, the end replication problem prevents telomeres from being fully replicated during cell division [51]. The telomerase enzyme is responsible for synthesizing the telomere, which is encoded by the telomerase reverse transcriptase (TERT) gene, which also contains a catalytic subunit and RNA template [52, 53]. Telomere shortening denotes the mitotic clock which is responsible for aging, but not in stem cells and germ cells [54, 55]. The expression of the genes located in the sub telomeric regions is often affected by the telomere shortening and it is denoted as “Telomere positioning effect” [56].

Without telomeres, chromosomes might end up adhering with each other [57]. In a young and healthy cell, the telomere sequence is usually guarded by a complex of proteins called shelterin complex or telosome. The telosome controls the length of the telomere and prevents end-to-end fusion by shielding it from DNA repair mechanisms that detect double-stranded breaks [58, 59]. The shelterin complex consists of six proteins; telomeric repeat-binding factor 1 (TRF1), telomeric repeat-binding factor 2 (TRF2), telomeric repeat-binding factor 1 interacting nuclear factor 2 (TIN2), repressor activator protein 1 (RAP1), tripeptidyl peptidase 1 (TPP1) and protection of telomere protein 1 (POT1) [60]. Major DNA damage signaling pathways such as non-homologous end joining (NHEJ) and homology-directed repairing get activated by misreading the telomeric ends as double-stranded DNA breaks. This misreading is done by ATM and ATR kinases, Homology-Directed Repair (HDR), which are typically blocked by shelterin complex in a healthy young cell [61]. Telomere sites become vulnerable to DNA damage and get easily recognized by repair proteins when shelterin’s protective function is lost. Apart from the above functions, the shelterin complex prevents telomerase binding as well which is usually involved in the telomere synthesis which results in immortality [62]. Some cells delay senescence and continue to proliferate by acquiring genetic mutations in p53 and other checkpoint proteins [63]. At this stage, a small proportion of telomerase active cells initiate the process of carcinogenesis [64].

Researchers are exploring strategies to selectively activate telomerase specifically in adult stem cells, rather than in somatic cells, to harness its regenerative potential while minimizing risks [65]. In a study, adeno-associated virus (AAV) vectors were utilized to deliver the TERT gene along with a promoter that specifically targets expression in stem cells or particular organs through intravenous administration. This regulated activation resulted in a 24% increase in lifespan for 1-year-old mice and a 13% increase for 2-year-old mice, without causing any instances of uncontrolled cellular proliferation [66]. In a distinct neurodegenerative study, AAV9-TERT vectors were utilized to transduce the TERT gene, which is specifically designed for targeting neuronal cells. The results revealed an upregulation of doublecortin, a protein that is exclusively expressed in immature neuronal cells, indicating that neuronal progenitor cells are differentiating into immature neurons. This evidence suggests that the AAV9-TERT vector successfully enhances telomerase expression, facilitating neuronal cell regeneration [67]. Several compounds have been identified as potential telomerase activators. One such compound is TA-65, which has been studied for its ability to activate telomerase and promote telomere elongation. Another compound, Cycloastragenol, has also been investigated for its capacity to activate telomerase and enhance cellular health, particularly in models related to female fertility and aging [68].

The term “microbiota” denotes a community of microorganisms that coexist in an ecosystem [69], while “microbiome” refers to a collection of all microorganisms, their genes, and their metabolites [70, 71]. Usually, microbiome inhabits in the respiratory tract, intestinal tract, urinary tract, skin, and majority of them colonize the colon [72, 73]. The microbiota is initially acquired during birth from the maternal birth canal and the composition of microbiota is comparatively different in the infants born through caesarean section [74, 75]. The majority of the human microbiota is made up of anaerobes; however, facultative anaerobes and aerobes coexist. More than 50 bacterial phyla exist in the human gut microbiota, among which two bacterial phyla, Bacteroidetes and Firmicutes, exist abundantly [76, 77].

The energy produced by the gut microbiota plays a role in energy-intensive processes such as cell growth and temperature homeostasis [78, 79]. A healthy gut with microbial derived metabolites (short-chain fatty acids [SCFAs]) provides sufficient energy to the cells of the intestinal epithelium and also enhances the integrity of the barrier by activating genes that regulate the critical balance between pro- and anti-inflammatory chemical messengers. The gut microbiome promotes lipid metabolism by elevating the expression of the enzyme cofactor co-lipase, which is required by pancreatic lipase to metabolize triglycerides into monoglycerides and fatty acids [80]. Dysbiosis is a consequence of variety of environmental factors, including food, toxins, medications and pathogens, predominantly enteric pathogens and results in detrimental diseases in humans, like gastric ulcer [77], irritable bowel syndrome [81], colorectal cancer [82], metabolic associated fatty liver disease [83], and obesity [84].

Several studies on senescence indicate that aging is frequently associated with its circadian rhythm pattern [40]. Mice that lack the oscillation of the BMAL1, PER1, PER2, and CLOCK proteins exhibit early signs of aging [9]. A study in genomic DNA of bipolar disorder (BD) patients revealed the leukocyte telomere length was found to be decreased in BD patients in comparison to healthy controls [85, 86]. A study in human smooth muscle cells reported that young and healthy human smooth muscle cells with stable and long telomeric structures can recover from circadian clock interference caused by serum shock (Fig. 3) [87].

A CLOCK^−/− and wild mice are entrained for 14 days in a 12 h dark/12 h light (LD) cycle. The telomerase activity in the liver was decreased in CLOCK^−/− mice in comparison to the wild type. Further, the length of the telomere was evaluated after the first passage of mouse embryonic fibroblast (MEF) cells showed that there was a decrease in telomere length in CLOCK^−/− MEF in contrast to wild type. The hTERT and mTERT promoters have E-boxes that CLOCK/BMAL1 can bind to, which are at −165 bp and −808 bp upstream of the transcription start site for each gene, respectively [21, 88].

The telomere repeat-containing RNA (TERRA) has been reported to get expressed in a diurnal rhythm, and it has been found to dampen with age. The H3K9me3 which maintains the repetitive elements and non-coding region of the genome exhibits diurnal rhythm. It could be hypothesized that the expression of H3K9me3 gets affected with aging as a result of a dysregulated circadian rhythm, and the expression of TERRA would likewise be disturbed. This proves conclusively that circadian rhythm disruption causes telomere shortening and occurs with aging [89].

Mammals have seven sirtuins that use NAD+ as a substrate to deacetylate proteins. The activity of SIRT1 deacetylase has been linked with chromatin remodeling, gene silencing, DNA damage response, apoptosis prevention, and indirect protection of telomere shortening. SIRT1^−/− mice and mice with additional copies of SIRT1, known as SIRT1^super mice, were employed in a study to investigate the SIRT1 relationship with telomere shortening. SIRT1 super animals were shown to have longer telomeres than SIRT1^−/− mice [90]. The expression of CLOCK/BMAL1 is positively correlated with the SIRT1 expression, indicating a possible transcriptional regulation of SIRT1. Therefore, the disrupted circadian rhythm could promote accelerated telomere shortening. SIRT1 is a crucial molecule in maintaining a stable telomeric length and a well-timed circadian clock [91, 92].

Gene expression related to sex differences is described by activation (hormone induced) of neuronal steroid receptors. This is observed in aging. Interaction of estrogens and androgens with their nuclear receptors (ER and AR) and the consequent binding of sex hormones with ER and AR entice epigenetic modifier enzymes along with corepressors, coactivators, and promoters of target genes. Once bound to its ligands, the nuclear hormonal receptors have an affinity to DNA sequences, referred to as hormonal response elements. This suggests that hormonal milieu formed by estrogens and androgens are efficient in altering age-related epigenetic mechanisms. This may consequently result in sexual differences in cognition, behavior, and specific immunological responses [93].

Light zeitgeber controls the central circadian clock and food zeitgeber regulates the intestine clock. The food zeitgeber also happens to regulate the circadian rhythm of exogenous components of the intestinal system, i.e., the gut microbiome. The circadian rhythm of the host can influence the composition of the gut microbiome and vice versa [92]. In a research study, stool sample was collected from a subject with flight induced jet lag from three different points of time; 24 h before jet lag, during jet lag and after recuperating (2 weeks) from jet lag. The microbial community in the stool sample obtained during jet lag showed a marked change in the composition of commensal microbiota with a distinct increase in firmicutes. Whereas the composition of the microbiota in the stool samples obtained before and after the jet lag was typical to that of a healthy human. The microbiota isolated from all the three samples was introduced into germ-free mice and the mice which received the microbiota from the stool sample obtained during jet lag exhibited a marked increase in fat accumulation [94]. A study in mice has demonstrated the disruption of circadian rhythm by mutating the core molecular clock (male clock^Δ19) with high fat diet and alcohol resulted in dysbiosis. The microbial community in faeces of the male clock^Δ19 mutant mice was analyzed and resulted in decreased taxonomical diversity in comparison with wild type [95].

A wide range of cells express pattern recognition receptors, they identify common molecular patterns shared by both commensal and pathogenic bacteria, and are referred to as pathogen-associated molecular patterns [96, 97]. TLR of IEC recognize microbiome metabolites and integrate the signal in to c-JUN terminal and IKKβ pathway, this prevents the activation of PPARγ, which in turn reduces expression of Rev-erbα [98, 99]. Typically, Rev-erbα, a Bmal1 repressor, and Rorα, a Bmal1 inducer, competes to bind to the E-box of Bmal1 gene. During dysbiosis the IEC receives dysregulated metabolic cues which leads to constitutive expression of Rev-erbα and eventually leads to dampening of Bmal1 oscillation [100]. Hence, any attempt to disrupt gut microbiome homeostasis, for instance through antibiotic treatments, can cause dysbiosis and disturbs circadian rhythm of the host IEC [101].

The circadian transcription factor NFIL3 is employed by the gut microbiome to regulate body composition. Additionally, the innate lymphoid cell-3 pathways of these cells are also responsible for transmitting signals from the microbiota to epithelial cells to regulate the transcription of NFIL3 [102].

There have been research studies conducted “hitherto” that demonstrate the link between microbiota and host epigenetic tackle. The dynamic axis between nutrient intake, neuroendocrine pathways, gut microbiome and physiology of the gut is interlaced. Their connection has substantial influence on epigenetic progressions involving cell cycle regulation, gene repair, gene expression, non-coding RNA activity, and DNA methylation [103].

Composition of gut microbiota changes in reaction to circadian rhythm [104]. Reduced mobility, impaired immune system, decreased function of the intestine, abnormal gut morphology and physiology, recurring infections, hospitalizations, and use of several medications contribute change in the gut microbiota [105, 106]. Studies have found that metformin increases the growth of the commensal bacteria and reduces enterobacteria. Also, the restoration of commensal bacteria enhances the production of SCFA, which maintains the intestinal barrier integrity, and controls the release of digestive peptides [107, 108]. The tight junctions of the IEC prevent the passage of microorganisms and undigested food particles into the systemic circulation [109].

A recent study observed that the nucleotide-binding oligomerisation domain-like receptor via the pathogen-associated molecular pattern of the microbiome can trigger inflammation once there is an imbalance in the gut microbiota (dysbiosis), leading to in a change in commensal bacteria and an increase in the flow of pathogenic bacteria [110]. Helicobacter pylori is believed to be a part of the normal gut microbiome and at the same time it is recognized as a causative agent of gastric cancer in several studies. Hence, the role of H. pylori being a beneficial microbe is debatable [111]. H. pylori can produce cyto-lethal distending toxin-like toxins which damage telomere and upregulate YAP1 transcriptional activity. YAP1 tends to upregulate inflammatory genes like pre-IL-1 β [112]. The IL-1β recruits T cells which in turn secretes IFN-γ, which instigates inflammation and oxidative stress causing telomere attrition in gastric cancer.

The telomere shortening and dysfunction is high during aging, due to decreased level of antioxidants and increased level of oxidative stress [113]. The gut microbiome generates reactive sulfur species (RSS) by metabolizing sulfur containing amino acids in the intestine, which possess high antioxidant activity [114]. RSS is produced by the host organs using enzyme cystathionine γ-lyase (CSE) and cystathionine β-synthetase (CBS) [115]; similarly, some bacteria of the gut microbiome also possess sulfur-metabolizing enzymes, like CSE and CBS [116]. Polyamines are organic polycationic alkylamines; they are synthesized by mammals in three forms such as spermidine, spermine, and putrescine [117, 118]. Polyamines upregulate the mucous secretion and induce the development of intact tight junctions, possess antioxidant properties, downregulate inflammatory cytokines and prevent inflammation [119]. Mice fed with PA-rich diet exhibited decreased incidence of age-related pathologies compared to mice fed with PA-poor diet have been reported [120]. Administration of probiotics in hospitalized patients revealed increased production of polyamines and reduced inflammatory markers [96].

Recent research has indicated that telomere, circadian rhythm, and gut microbiome are all bilaterally linked (Fig. 4). The disruption of the circadian rhythm will lead to premature senescence by promoting telomere attrition and increase the population of pathogenic bacteria in the gut which disrupts the integrity of intestinal niche [121]. Improper sleep, unbalanced diet, aberrant meal time and mental disorders drive aging by disrupting the circadian rhythm. The circadian rhythm controls the telomere length directly by regulating SIRT1 [89, 122], CLOCK/BMAL1 [123], and c-Myc [94] and indirectly by controlling the composition of the gut microbiome and their microbial diversity. Mice that were consistently fed throughout the day for a week showed a significantly altered circadian expression profile. The dysbiosis caused by the disrupted circadian rhythm leads to the decreased production of antioxidants such as RSS and polyamines, which usually decreases the oxidative stress in the IEC. The antioxidant defense fails due to lower composition of commensal bacteria in case of a disrupted circadian rhythm. The use of antibiotics typically causes dysbiosis, and it can lead to the disruption of circadian rhythm, while induction of prebiotics leads to the increase in commensal bacterial population in the gut which rewires the clock [124]. Circadian rhythms can also be altered by metabolites produced by the microbiota, such as SCFA and bile acids [125, 126]. Apart from this, the senescent IEC contributes to the senescence-associated secretory phenotype that instigates recruitment of immune cells, which causes intestinal inflammation and gut dysbiosis. Pathogenic bacteria like H. pylori, Enterobacter spp. drives inflammation by activating host TLR receptors which causes phagocytic cell derived oxidative stress. This oxidative stress is very damaging to the telomere thus promoting cellular senescence which has already been known to disrupt the circadian clock. A well-oiled circadian clock machinery corresponds to a stable telomere biology.

The interconnection of the circadian rhythm, gut microbiota, and telomere explains how each of them is strongly interrelated and loss of homeostasis in one can influence the others. Also, the connection between circadian rhythm, gut microbiota, and epigenetic mechanisms is noteworthy. The interplay between sex differences and epigenetic regulation along with role of telomere cannot be ruled out. Longevity in vertebrate systems is fully functional through a healthy lifestyle. This emphasizes and concludes on the role of biological clock, nutrient intake, on molecular mechanisms.

The authors have no conflict of interest to declare.

No funding was received.

Anup Kumar Mani and Sumitha Ravindran drafted and revised the manuscript. Anup Kumar Mani worked on the illustrations as well. Venkatachalam Deepa Parvathi supported in designing the study, reviewed, revised, and edited the manuscript. All authors have read and approved the final manuscript.

Data availability is not applicable as this is a review article. However, data associated with the review are available on request from the corresponding author.