Circadian Clock NAD+ Cycle Drives Mitochondrial Oxidative Metabolism in Mice

Introduction The circadian clock is a transcriptional oscillator that is thought to couple internal energetic processes with the solar cycle. Circadian oscillation in activity of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD+) biosynthesis, feeds back to regulate activity of the deacetylase SIRT1 and transcription of genes encoding core clock components. Despite evidence that NAD+-dependent enzymes are important in fasting and oxidative metabolism, it is not known how the circadian cycle might affect this process. We investigated the role of clock control of NAD+ in mitochondrial dynamics and energy production. Circadian regulation of NAD+ biosynthesis synchronizes mitochondrial bioenergetics with the light-dark cycle. The core molecular clock is a transcription-translation oscillator composed of activators (CLOCK/BMAL1) that induce transcription of their own repressors (PER/CRY). Clock control of expression of the NAD+ biosynthetic enzyme NAMPT generates 24-hour variation of activity of the mitochondrial deacetylase SIRT3 and oxygen consumption. Rhythmic NAD+ oscillation couples mitochondrial bioenergetics with the light-dark cycle. Methods We determined the circadian variation in mitochondrial function by examining the adaptive response to fasting in liver of wild-type and circadian mutant mice. Quantitative analyses of NAD+ biosynthesis, lipid and glucose oxidation, and acetylation of mitochondrial proteins were performed across the circadian cycle in circadian mutant mice and in cell-based systems. Proteins displaying increased acetylation in Bmal1 mutant liver were identified by mass spectrometry, and SIRT3 activity was evaluated using label-free self-assembled monolayer and matrix desorption ionization (SAMDI) mass spectrometry in liver lysate from Bmal1 and Sirt3 knockout mice. The role of NAD+ deficiency in SIRT3 activity, mitochondrial protein acetylation, lipid oxidation, and oxygen consumption was evaluated after intraperitoneal administration of the NAD+ precursor NMN to raise NAD+ levels in Bmal1 mutant and wild-type mice. Results Lipid oxidation and mitochondrial protein acetylation exhibited circadian oscillations that corresponded with the clock-driven NAD+ cycle in mouse liver. Rhythmic NAD+ and oxidative cycles were self-sustained in fasted mice and in C2C12 myotubes, demonstrating clock control of mitochondrial function even when nutrient state remained constant. Transcription of glycolytic genes was antiphasic to lipid oxidation rhythms, and glycolytic gene expression and lactate production were increased in Bmal1–/– fibroblasts, whereas the converse occurred in Cry1–/–;Cry2–/– mutants. Lack of Bmal1 in liver led to decreased SIRT3 activity and increased mitochondrial protein acetylation, resulting in reduced function of oxidative enzymes. Finally, NAD+ supplementation with NMN restored protein deacetylation of SIRT3 targets and enhanced mitochondrial function in circadian mutant mice. Discussion Mitochondria are central to energy homeostasis in eukaryotes, and our results show that the circadian clock generates oscillations in mitochondrial oxidative capacity through rhythmic regulation of NAD+ biosynthesis. The clock thereby facilitates oxidative rhythms that correspond with the fasting-feeding cycle to maximize energy production during rest. Use of NAD+ as a central node in coupling circadian and metabolic cycles provides a rapid and reversible mechanism to augment mitochondrial oxidative function at the appropriate time in the light-dark cycle. Dinner Time! Biological clocks allow organisms to anticipate cycles of feeding, activity, and rest so that metabolic enzymes in mitochondria are ready when needed. Peek et al. (10.1126/science.1243417, published online 19 September; see the Perspective by Rey and Reddy) describe a mechanism by which the biochemical elements of the circadian clock are linked to such control of mitochondrial metabolism. The clock controls rhythmic transcription of the gene encoding the rate-limiting enzyme required for synthesis of nicotinamide adenine dinucleotide (NAD+). The concentration of NAD+ in mitochondria determines the activity of the deacetylase SIRT3, which then controls acetylation and activity of key metabolic enzymes. NAD+ also influences clock function, and thus appears to be a versatile point at which regulation of oxidative metabolism is coordinated with the daily cycles of energy consumption. The coenzyme nicotinamide adenine dinucleotide mechanistically links the circadian clock to control of energy production by mitochondria. [Also see Perspective by Rey and Reddy] Circadian clocks are self-sustained cellular oscillators that synchronize oxidative and reductive cycles in anticipation of the solar cycle. We found that the clock transcription feedback loop produces cycles of nicotinamide adenine dinucleotide (NAD+) biosynthesis, adenosine triphosphate production, and mitochondrial respiration through modulation of mitochondrial protein acetylation to synchronize oxidative metabolic pathways with the 24-hour fasting and feeding cycle. Circadian control of the activity of the NAD+-dependent deacetylase sirtuin 3 (SIRT3) generated rhythms in the acetylation and activity of oxidative enzymes and respiration in isolated mitochondria, and NAD+ supplementation restored protein deacetylation and enhanced oxygen consumption in circadian mutant mice. Thus, circadian control of NAD+ bioavailability modulates mitochondrial oxidative function and organismal metabolism across the daily cycles of fasting and feeding.