Circadian rhythms are endogenous oscillations that coordinate the physiological and behavioral processes with the 24-hour cycle of day and night [1]. Circadian rhythms are controlled by a master clock network, comprising the paired suprachiasmatic nuclei in the hypothalamus, and the pineal gland via the 24-hour regulation of melatonin synthesis and secretion [2]. Disruptions in circadian rhythmicity have been implicated in various pathological conditions, including metabolic disorders [3], cardiovascular diseases [4], neurodegeneration [5], and cancer [6]. Understanding the molecular mechanisms underlying circadian rhythm regulation is crucial to develop targeted therapeutic interventions [7]. Nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in redox reactions and substrate for various enzymes, has recently emerged as a key metabolite in the regulation of circadian rhythms [8]. NAD+ levels oscillate in a circadian manner, and several NAD+-dependent enzymes have been shown to modulate the expression and activity of the core clock components [9]. Furthermore, the biosynthesis of NAD+ is influenced by the circadian clock machinery, which creates a bidirectional regulatory loop [10]. Sirtuins, a family of NAD+-dependent protein deacetylases, have garnered significant attention for their role in regulating various cellular processes, including metabolism, stress responses, and aging [11,12]. Emerging evidence suggests that sirtuins are intimately linked to the circadian clock, acting as both upstream regulators and downstream effectors of the circadian machinery [13]. Moreover, sirtuin (SIRT) 1, a mammalian sirtuin, deacetylates and modulates the activity of key circadian transcriptional regulators, such as brain and muscle arnt-like 1 (BMAL1) and period circadian regulator 2 (PER2) [14,15]. Additionally, SIRT6 has been implicated in the circadian control of metabolism through the regulation of glycolytic and gluconeogenic pathways [16]. This mini-review aims to provide a comprehensive overview of the relationships among NAD+, sirtuins, and the circadian clock machinery. Furthermore, it explores the mechanisms by which NAD+ biosynthesis and degradation pathways are regulated by the circadian clock and the role of sirtuins as both sensors and effectors of circadian rhythms. The effects of circadian disruption on NAD+ homeostasis and sirtuin activity are discussed, focusing on the potential contributions of circadian disruption to aging and age-related disorders. The development of NAD+ boosters and sirtuin modulators could be a potential therapeutic strategy for restoring circadian rhythmicity and promoting healthy aging.

Numerous studies have established a bidirectional relationship between NAD+ and the circadian clock machinery. NAD+ levels exhibit robust circadian oscillations in various tissues, including the liver, skeletal muscle, and brain [17-19]. These oscillations are driven by the rhythmic expression and activity of enzymes involved in the NAD+ biosynthesis and degradation pathways (Fig. 1). NAD+ serves as an essential cofactor for several enzymes that directly interact with the core clock components, thereby influencing circadian rhythmicity [20]. Although poly(adenosine diphosphate [ADP] ribose) transferase is involved in NAD+-consuming pathways, it does not possess deacetylase activity [21]. Poly(ADP-ribose) polymerase (PARP), which catalyzes the transfer of ADP-ribose units from NAD+ to target proteins, modulates the activity of circadian transcriptional regulators such as circadian locomotor output cycles kaput (CLOCK) and BMAL1 through ADP-ribosylation [22].

NAD+ biosynthesis is regulated by the circadian clock machinery, which further highlights the relationship between the two systems. Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in this process that catalyzes the rate-limiting step in the NAD+ salvage pathway [23]. NAMPT expression and activity exhibit circadian oscillations, which are driven by the core clock components CLOCK and BMAL1 [24]. Additionally, the circadian clock regulates the expression of other enzymes involved in NAD+ biosynthesis, such as nicotinamide mononucleotide adenylyl transferase (NMNAT) and nicotinic acid phosphoribosyl transferase [25]. Conversely, NAD+ levels and the metabolic state of NAD+ influence the activity of the clock components. For instance, the NAD+-dependent deacetylase SIRT1 deacetylates and modulates the activity of BMAL1 and PER2, thereby influencing the circadian transcriptional-translational feedback loop [26]. Furthermore, PARP-mediated ADP-ribosylation of CLOCK and BMAL1 can alter their transcriptional activity and stability, respectively [27].

Sirtuins are a conserved family of NAD+-dependent protein deacetylases that catalyze the removal of acetyl, succinyl, malonyl, and other acyl groups from target proteins (Fig. 2). Mammals possess seven sirtuin isoforms (SIRT1-SIRT7) that differ in their subcellular localization, enzymatic activity, and substrate specificity [28]. SIRT1, the most extensively studied mammalian sirtuin, is a nuclear and cytoplasmic deacetylase that plays crucial roles in various processes, such as energy balance and regulation of circadian rhythms in the hypothalamic neurons. The disruption of this pathway is closely linked to the pathogenesis of obesity and aging [29]. SIRT2, predominantly localized in the cytoplasm, deacetylates cytoskeletal proteins and regulates cell cycle progression [30]. SIRT3, SIRT4, and SIRT5 are mitochondrial sirtuins involved in regulating mitochondrial metabolism and redox homeostasis [31]. SIRT6, a nuclear sirtuin, has been implicated in DNA repair, telomere maintenance, and metabolic regulation [32]. SIRT7, another nuclear sirtuin, interacts with RNA polymerase I and regulates ribosomal biogenesis [33]. SIRT6 has been implicated in the circadian regulation of metabolism through its interactions with key metabolic regulators. SIRT6 deacetylates and modulates the activity of histone H3 (at lysine 9 and 56), thereby regulating the expression of circadian-controlled metabolic genes involved in glycolysis, gluconeogenesis, and lipid metabolism [34]. Additionally, SIRT6 interacts with and deacetylates the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha, a key regulator of mitochondrial biogenesis and function [35]. While SIRT1 and SIRT6 are the most extensively studied sirtuins in the context of circadian rhythms, other sirtuin isoforms have also been implicated in circadian regulation. For instance, SIRT3 influences the circadian rhythms in mitochondrial respiration and adenosine triphosphate production [36].

Accumulating evidence suggests a bidirectional relationship between circadian disruptions and alterations in NAD+ homeostasis [37]. Circadian misalignment induced by conditions, such as shift work, jet lag, or exposure to irregular light-dark cycles, can lead to dysregulation of NAD+ biosynthetic enzymes, including NAMPT and NMNAT [38,39]. Conversely, fluctuations in NAD+ levels can affect and disrupt the molecular mechanisms of the circadian clock [40]. Depletion of NAD+, either through genetic manipulation of biosynthetic enzymes or pharmacological inhibition, can lead to altered expression and activity of the core clock components, such as BMAL1 and CLOCK [41]. These effects are mediated, at least partly, by NAD+-dependent enzymes, including sirtuins and PARPs, which modulate the acetylation and ADP-ribosylation of clock proteins. Decreased NAD+ levels are a hallmark of aging in various organisms including mammals [42]. This age-associated NAD+ depletion has been attributed to several factors, including decreased expression of biosynthetic enzymes (e.g., NAMPT), increased activity of NAD+ consumers (e.g., PARPs), and impaired salvage pathways [43]. A reduction in NAD+ levels during aging has profound consequences on sirtuin activity and function. As NAD+-dependent enzymes, sirtuins require sufficient NAD+ availability to perform deacetylation activities [44]. Reduced SIRT1 activity is associated with impaired glucose and lipid metabolism, which contributes to the development of insulin resistance and obesity [45,46]. Similarly, SIRT6 deficiency is associated with accelerated aging and age-related pathologies, including genomic instability and cancer [47]. Given the relationships among NAD+, sirtuins, and the circadian clock, strategies aimed at restoring NAD+ levels or modulating sirtuin activity have emerged as potential therapeutic strategies for circadian rhythm disorders and age-related pathologies (Fig. 3) [48].

Approaches to boost NAD+ levels, such as supplementation with NAD+ precursors (e.g., nicotinamide riboside [NR] and nicotinamide mononucleotide [NMN]) or inhibition of NAD+ consumers (e.g., PARP inhibitors), can restore circadian rhythmicity and ameliorate age-related phenotypes in preclinical models [49,50]. Additionally, small-molecule sirtuin activators or inhibitors have been developed and explored for their potential to modulate circadian rhythms and associated metabolic processes [51].

The association between NAD+, sirtuins, and the circadian clock has spurred intense research efforts to develop and characterize novel NAD+ boosters and sirtuin modulators. These compounds are potential therapeutic agents for circadian rhythm disorders and age-related pathologies associated with NAD+ decline and sirtuin dysfunction [52]. Several NAD+ precursors, such as NR and NMN, have been explored for their ability to increase intracellular NAD+ levels and modulate circadian rhythms. NR supplementation restored circadian rhythmicity and ameliorated age-related metabolic dysregulation in animal models. 53 Short-term supplementation of NMN in pre-aging mice and humans aged 45–60 years showed a significant increase in the telomere length in peripheral blood mononuclear cells and altered fecal microbiota, benefiting immune and metabolic pathways [54]. In addition to NAD+ precursors, inhibitors of NAD+ consumers, such as PARP inhibitors, have attracted increasing interest as potential circadian modulators. PARP inhibition increased NAD+ availability, enhanced sirtuin activity, and restored circadian rhythmicity in various experimental models [55]. Regarding sirtuin modulation, small-molecule activators and inhibitors have been developed and investigated for their effects on circadian rhythms and associated physiological processes. For instance, selective SIRT1 activators modulated circadian rhythms and metabolic homeostasis in animal models [56]. Conversely, SIRT1 inhibitors have been explored for their potential to synchronize circadian rhythms and modulate sleep-wake cycles [57]. Circadian oscillations in NAD+ levels and sirtuin activities suggest that the timing of therapeutic interventions targeting these pathways may be crucial for optimal efficacy and minimizing off-target effects. The emerging field of chrono-pharmacology explores the concept of optimizing drug delivery and dosing schedules based on circadian rhythms and biological times [58]. Several studies have investigated the timed delivery of NAD+ precursors, such as NMN, to maximize their effects on circadian rhythms and metabolic processes. For example, time-restricted administration of NMN enhanced circadian rhythmicity and metabolic homeostasis in animal models of circadian disruption and aging [59]. Furthermore, the development of controlled-release or sustained-release formulations of NAD+ precursors may enable more stable and sustained modulation of NAD+ levels and sirtuin activities, potentially leading to improved circadian rhythm regulation and therapeutic outcomes [60].

This review highlights the relationships among NAD+, sirtuins, and the circadian clock machinery. NAD+ levels exhibit robust circadian oscillations that are driven by the rhythmic expression and activity of biosynthetic enzymes such as NAMPT. Conversely, the circadian clock regulates the expression of various NAD+-biosynthetic enzymes, creating a bidirectional regulatory loop. Sirtuins, a family of NAD+-dependent deacetylases, play crucial roles in modulating the circadian clock by interacting with the core clock components and transcriptional regulators. Furthermore, SIRT1 and SIRT6 have emerged as key regulators of circadian rhythms and metabolic homeostasis. Although there has been substantial progress in elucidating the mechanisms linking NAD+, sirtuins, and circadian rhythms, several challenges and limitations remain. The tissue- and isoform-specific functions of sirtuins and their complex relationships with other cellular pathways make it difficult to predict the systemic effects of sirtuin modulation. Additionally, the feasibility and safety of long-term NAD+-boosting strategies should be carefully evaluated, as chronic elevation of NAD+ levels may have unintended consequences. The findings of this review have important implications for developing circadian medicine and interventions that promote healthy aging. Strategies aimed at restoring NAD+ levels or modulating sirtuin activity may have potential in treating circadian rhythm disorders and age-related pathologies associated with circadian dysregulation. However, a deeper understanding of tissue-specific and temporal aspects of NAD+ and sirtuin biology is crucial for developing targeted and effective therapeutic approaches. Furthermore, the links among NAD+, sirtuins, and circadian rhythms may provide insights into the molecular mechanisms underlying aging and age-related diseases. Targeting these pathways may offer novel opportunities for developing interventions that not only restore circadian rhythmicity but also delay the onset and progression of age-related pathologies associated with NAD+ decline and sirtuin dysfunction. Future research should focus on further elucidating the complex regulatory networks linking NAD+, sirtuins, and the circadian clock machinery, as well as exploring the potential of chrono-pharmacological approaches and epigenetic modulation strategies. Additionally, interdisciplinary collaborations between circadian biologists, aging researchers, and clinicians are crucial for translating these findings into effective therapeutic interventions for circadian rhythm disorders and age-related pathologies.