Circadian rhythms—physical, mental, and behavioral changes that follow a daily cycle, such as the sleep-wake cycle—get disrupted as we get older, which can contribute to the development of diseases associated with aging. However, the mechanisms and signals that link circadian rhythms and aging are unclear.

A research team from Northwestern University published an article in the journal Molecular Cell showing that the molecule nicotinamide adenine dinucleotide (NAD+) controls global circadian rhythm signals and can prevent age-related decline in circadian function in mice. They found that raising NAD+ levels restores youthful gene activation and rhythms of activity in the cell’s powerhouse, the mitochondria, in old age. These findings reveal that NAD+ has a major role in affecting both behavior and metabolic health.
Raising nicotinamide adenine dinucleotide (NAD+) restores youthful gene activation and mitochondrial rhythms in old age. NAD+ induces the activation of genes related to stress and metabolism through the circadian clock. The protein BMAL1 is required for the response to NAD+. NAD+ regulates PER2 localization through acetylation to control gene activation related to circadian rhythms. NAD+ counters age-related decline in circadian function. P, phosphorylation; ac, acetylation (Levine Molecular Cell | 2020).

The levels of NAD+ in our cells decline as we age. However, it has been shown that boosting NAD+ production by administering its precursors promotes youth in mice. NAD+ is a molecule necessary for the function of sirtuins—enzymes known for promoting healthspan and lifespan. A member of this family of enzymes called Sirtuin 1 (SIRT1) regulates circadian rhythms by binding to members of the “core clock complex,” which consists of gene activators CLOCK–BMAL1 and the gene repressor PER2. To do so, SIRT1 modifies PER2 by a process called deacetylation, which leads to the degradation of PER2 and activation of certain genes related to the circadian rhythm.

To investigate the role of NAD+ in circadian gene expression in mice, the researchers gave mice drinking water supplemented with NAD+ precursors, specifically nicotinamide riboside (NR), for 4 months and then analyzed how genes related to circadian rhythms were activated in the liver. This revealed that the activation pattern of roughly half the circadian-regulated liver genes changed upon increasing NAD+. “Our observation that NR supplementation exerts broad effects on rhythmic gene transcription in liver and our identification of NAD+ as a regulator of SIRT1-mediated deacetylation of PER2 establishes a mechanism by which NAD+ regulates the clock,” said the investigators.

When the investigators administered NR and increased NAD+ levels, it enhanced the function of the circadian gene activator BMAL1 in mouse livers and, ultimately, increased the circadian rhythm gene activation. On the contrary, when the investigators eliminated SIRT1 from the liver in mice, it abolished the changes in circadian rhythm gene activation in the liver that were observed when NAD+ levels were increased. This showed increased NAD+ levels improve circadian rhythm through a SIRT1-dependent mechanism.

Moreover, the localization of PER2 in the nucleus, which is where it acts to repress genes linked to circadian rhythms, was increased in cultured cells deficient for SIRT1. Also, when the investigators depleted NAD+ in mice, they observed increased modifications of PER2 that promote its stability and activity as well as its retention in the nucleus and, therefore, its functionality. Altogether, NAD+ curbs PER2 activity as a circadian gene repressor, thereby increasing circadian gene activation through BMAL1. This shows that NAD+ promotes gene activity controlled by BMAL1 through SIRT1 and PER2.

Next, the investigators found that the function of BMAL1 in the liver of old mice was decreased, which coincided with increased PER2 levels and reduced the intensity of circadian gene oscillations. When the investigators administered the NAD+ precursor NR to the old mice for 6 months, they observed restored BMAL1 function to levels similar in young animals.

Nicotinamide riboside (NR) enhances circadian gene activation patterns and metabolic function in old mice. Left: average oscillations of gene activation from young and old water- and NR-treated mice. Right: Oxygen consumption rates (OCR) of the cell’s powerhouse (i.e., mitochondria) isolated from young and old, water- and NR-treated liver (Levine Molecular Cell | 2020).

Also, when they administered NR, the amount of late-night physical movement, which is normally reduced in old mice, was restored to youthful levels. “Provision of NR…in drinking water of old mice countered the decline in nighttime locomotor activity rhythms,” said the investigators.

Nicotinamide riboside (NR) restores evening locomotor activity in old mice. Left: representative average 24-hour profile of wheel-running activity of young and old water- and NR-treated mice. Arrow denotes ‘‘late-night activity.’’ Right: quantification of average wheel revolutions per minute in the late night (Levine Molecular Cell | 2020).

In summary, elevating NAD+ can reverse aging-associated dysfunction of circadian rhythms. Correction of circadian rhythmicity with NAD+ could also be tested for the management of circadian disorders linked to PER2 deregulation, such as those caused by PER2 mutations or shift work. “Our studies clarify the mechanisms underlying SIRT1-mediated regulation of the clock, for which discrepant mechanisms were previously proposed,” said the investigators.