Research Article: Altered behavioral and metabolic circadian rhythms in mice with disrupted NAD+ oscillation

Date Published: August 31, 2011

Publisher: Impact Journals LLC

Author(s): Saurabh Sahar, Veronica Nin, Maria Thereza Barbosa, Eduardo Nunes Chini, Paolo Sassone-Corsi.



The Intracellular levels of nicotinamide adenine dinucleotide (NAD+) are rhythmic and controlled by the circadian clock. However, whether NAD+ oscillation in turn contributes to circadian physiology is not fully understood. To address this question we analyzed mice mutated for the NAD+ hydrolase CD38. We found that rhythmicity of NAD+ was altered in the CD38-deficient mice. The high, chronic levels of NAD+ results in several anomalies in circadian behavior and metabolism. CD38-null mice display a shortened period length of locomotor activity and alteration in the rest-activity rhythm. Several clock genes and, interestingly, genes involved in amino acid metabolism were deregulated in CD38-null livers. Metabolomic analysis identified alterations in the circadian levels of several amino acids, specifically tryptophan levels were reduced in the CD38-null mice at a circadian time paralleling with elevated NAD+ levels. Thus, CD38 contributes to behavioral and metabolic circadian rhythms and altered NAD+ levels influence the circadian clock.

Partial Text

Circadian rhythms occur with a periodicity of about 24 hours and regulate a wide array of metabolic and physiologic functions. A robust circadian clock allows organisms to anticipate environmental changes and to adapt their behavior and physiology to the appropriate time of day. Disturbances in the functionality of this “body clock” have been shown to lead to various diseases, such as sleep disorders, depression, metabolic syndrome and cancer [1,2]. Circadian rhythms are regulated by transcriptional and post-translational feedback loops generated by a set of interplaying clock proteins. The transcription factors CLOCK and BMAL1 operate as the master regulators of the clock machinery. CLOCK:BMAL1 heterodimers bind to promoters of clock controlled genes (CCGs) and regulate their expression. Some CCGs are special in the sense that they encode other core-clock regulators, such as Period and Cryptochrome genes, which negatively feedback on the clock machinery [1-3]. Recently, the deacetylase sirtuin 1 (SIRT1) was identified as a modulator of the circadian clock machinery that counterbalances the acetyltransferase activity of CLOCK [4-7]. Moreover, an additional novel transcriptional/enzymatic feedback loop that regulates the circadian clock has recently been uncovered [8,9]. The circadian clock controls the levels of NAD+ by regulating the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis [8,9]. NAD+ is also synthesized from the amino acid tryptophan by the de novo synthesis pathway. Tryptophan levels have been shown to oscillate in plasma [10], perhaps contributing to the oscillations in NAD+ levels. NAD+ is a cofactor and /or substrate for over 300 enzymes and acts as a cellular energy currency. SIRT1 is one such enzyme whose deacetylase activity is NAD+-dependent [11]. In addition, poly(ADP-ribose) polymerase (PARP)-1 and PARP-2 are NAD+-consuming enzymes, and their deletion raises NAD+ levels in mice [12,13]. Moreover, the SIRT1 and PARPs systems have been linked as they possibly use the same NAD+ cellular pool [14]. Circadian oscillations in NAD+ levels drive SIRT1 rhythmic activity [4]. SIRT1, in turn, is recruited to the Nampt promoter along with CLOCK and BMAL1. Thus, the circadian machinery is regulated by an enzymatic/transcriptional feedback loop, wherein SIRT1 regulates the levels of its own coenzyme [8,9]. Interestingly, PARP-1 activity has also been shown to display circadian oscillation [15]. These findings highlight the intimate connections between the circadian clock and cellular metabolism [16].

Altered circadian NAD+ levels in CD38-null mice. It has been reported that the CD38-KO mice have elevated levels of NAD+ in most tissues [17,18]. We analyzed the profile of NAD+ levels throughout the circadian cycle. NAD+ levels were measured at various zeitgeber times (ZTs) and were found to oscillate in the liver as described [9] (Fig. 1A). The oscillation of NAD+ was significantly altered in the liver of CD38-KO mice, being much higher than in WT mice at ZT7 and ZT15, with unchanged levels at ZT23 (Fig. 1A). Most remarkably, NAD+ levels in the CD38-KO mice were about 5 times higher than in WT animals at ZT15. Next, we wanted to determine whether the differences in NAD+ levels between WT and CD38-KO mice may be due to a change in NADase activity. Indeed, NADase activity in the liver of CD38-KO mice was drastically lower as compared to the WT animals at ZT7 and ZT15 (Fig 1B; ref. 13). Surprisingly, CD38-KO mice display high level of NADase activity (~50% of the WT activity) at ZT23. While the reasons for this could be due to a time-specific increase in the expression/activity of another unrecognized NAD+ glycohydrolase, this result explains the comparable NAD+ levels at ZT23.

Our current work has addressed the question whether alterations in NAD+ rhythmicity in vivo could affect the functioning of central and peripheral clocks. For this purpose we explored the circadian behavior of CD38-KO mice which have elevated NAD+ levels. We observed that oscillations in NAD+ levels were altered in the liver of CD38-KO mice. Strikingly, at ZT 15 (the trough of NAD+ levels in WT liver), NAD+ levels peaked in CD38-KO mice and were ~ 5 times higher than those in the liver of WT mice. Surprisingly, NAD+ levels were similar between WT and CD38-KO mice at ZT23. This could be due to an increase at this circadian time of NADase activity in CD38-null mice. Although CD38 is the major NAD+ hydrolase in cells, it is possible that in its absence other NAD+ hydrolases (such as CD157, a gene duplication product of CD38) could be induced as a compensatory mechanism.