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

Abstract

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.

Figure 1. Effect of CD38 on NAD⁺ levels

WT and CD38 KO mice entrained in 12 hr Light - 12 hr Dark (LD) cycles were sacrificed at indicated times and their liver was dissected out. (A) NAD⁺ concentration was measured by a cycling enzymatic assay. *, p<0.05 (WT vs KO ZT7); **, p<0.001 (WT vs KO ZT 15) [n=3 each time point] (B) NADase activity was measured by a flurometric assay. **, p<0.001 (WT vs KO for each time point) [n=3 each time point].

Figure 2. Circadian defects in the behavioral rhythm of CD38-KO mice

(A) Representative activity records (actograms) of Wild type (CD38^+/+) and CD38 knockout (CD38^-/-) mice are shown in double plotted format. Mice were entrained in 12 hr Light - 12 hr Dark cycles (LD) and then placed in constant darkness (DD) from the light off (ZT12), on day 1. (B) Bar graph representing the period length of WT and CD38-KO mice. Measurement of the free-running period was based on the onset of activity in DD. Data is represented as mean ± S.E. * *, p = 0.008, n= 6, 8. (C) Representative actograms of Wild type (CD38^+/+) and CD38 knockout (CD38^-/-) mice in LD cycle. (D) Bar graph representing % daily locomotor activity in a one-hour period at the indicated ZTs. Data represents mean ± S.E of 10 days of activity. *, p < 0.05; **, p<0.01 compared to the corresponding wild type, n= 6, 8. (E) Representative actograms from wheel running activity of Wild type (CD38^+/+) and CD38 knockout (CD38^-/-) mice in LD cycle. (F) Bar-graph representing total number of wheel rotations per day. Data is represented as mean ± S.E. **, p = 0.008, n= 6, 5.

Figure 3. Differential liver gene expression in CD38-null mice

Mice entrained in 12 hr Light - 12 hr Dark cycles were sacrificed at indicated times and their liver was dissected out.(A) RNA was prepared at indicated times, reverse transcribed, and real-time PCR was performed using primers for Dbp, Per2, Nampt and 18S rRNA. Data is represented as relative levels of indicated gene normalized to 18S rRNA.(B) Same as in (A), except real-time PCR was performed using primers for Asns, Igfbp1, Moxd1 and c -Myc. Data is represented as relative levels of indicated gene normalized to 18S rRNA. *, p<0.05 compared to the corresponding wild type, n= 3 each time point.

Figure 4. Alterations in the plasma amino acid levels in CD38-KO mice

Mice Mice were entrained in 12 hr Light - 12 hr Dark cycles and blood was drawn at indicated times. Amino acid levels in plasma were determined as described in Materials and Methods. Amino acids that displayed statistically significant differences in abundance between the wild type and the CD38-KO mice are shown here. *, p<0.05; **, p<0.01 compared to the corresponding wild type, n= 3 each time point.