Circadian SCN-Liver Axis in the Neuroendocrine Response to Calorie Restriction

In mammals, the hypothalamic pacemaker clock synchronizes peripheral tissue clocks to temporally partition oxidative and reductive metabolic pathways to align fuel utilization with nutrient availability. Yet how the circadian clock in brain and peripheral tissues integrates nutrient state with transcription to promote energy conservation and metabolic homeostasis during sleep and in nutrient scarce conditions remains obscure. An exciting clue as to how nutrient signals control circadian transcription emerged from the discovery in our group and others that nicotinamide adenine dinucleotide (NAD+) and the NAD+-dependent deacetylase SIRT1 regulate circadian behavioral and mitochondrial rhythms through posttranslational modification of the core clock repressor PER2, indicating that NAD+-SIRT1 controls clock cycles within both neurons and peripheral cells. Interconversion of NAD+ with its reduced form NADH during redox reactions is dependent upon nutrient state. In new results published after our first submission, we show that NADH accumulation in liver during healthful calorie restriction inhibits SIRT1 and reduces daytime body temperature and oxidative metabolism. Surprisingly, reducing NADH levels through hepatic transduction of the water-forming NADH oxidase Lactobacillus brevis (LbNOX) disinhibits SIRT1 and augments oxidative cycles of metabolism and transcription. Further, our newly-generated PER2K680Q acetyl-mimetic knockin mice, which are resistant to SIRT1-induced deacetylation, exhibit profound period lengthening, while clock ablation in the suprachiasmatic nucleus (SCN) abrogates rhythmic feeding and thermogenesis. We are now poised with innovative genetic tools and circadian protocols to dissect how the circadian clock promotes energy constancy during sleep and in adaptation to calorie restriction at the level of the liver (Aim 1) and hypothalamic pacemaker neurons (Aim 2). Aim 1 will specifically test the hypothesis that nutrient sensing by the clock involves NAD(H)-SIRT1 signaling. We propose to dissect the role of redox state in clock function and metabolism during sleep and calorie restriction by genetically manipulating NAD(H) levels using LbNox in combination with hepatic clock ablation or PER2K680Q acetyl-mimetic knockin mice. Aim 2 will examine the role of hypothalamic pacemaker neuron subtypes in synchronizing thermogenesis, feeding, and metabolic rhythms with sleep and in the adaptive response to calorie restriction by utilizing an innovative combination of CRISPR-Cas9 clock ablation, loss and gain of function studies, and projection-based chemogenetic manipulation of pacemaker neurons. Collectively, our proposed studies will elucidate circadian mechanisms involved in maintenance of energy constancy across the sleep-wake cycle and how clock adaptations contribute to health benefits of hypocaloric diet.