PROJECT SUMMARY / ABSTRACT The 24/7 lifestyle in modern society disrupts natural circadian rhythms and poses a serious health concern. Epidemiological and laboratory evidence indicates that circadian disturbances adversely affect various biological functions and increase disease risk. The circadian clock, a network of cellular oscillators, is the biological timer driving output gene expression and physiological functions with ~24h rhythmicity. During aging, the clock and clock-controlled rhythms display attenuated oscillatory amplitude, concomitant with physiological and behavioral decline. Importantly, environmental/dietary interventions support a positive modifiable function of the clock to promote healthy aging. For example, several dietary interventions that prolong healthy lifespan and/or longevity, including caloric restriction and time-restricted feeding in the active phase (TRF), were found to enhance circadian gene oscillation and output metabolism. Together with genetic evidence, these observations strongly suggest a crucial regulatory role of robust circadian oscillation in healthy aging. We previously identified Nobiletin (NOB), a polymethoxylated dietary flavonoid, as a clock-enhancing compound. We found that NOB activates RORs (specifically the alpha and gamma subtypes, encoded by Rora and Rorc), key components of the cellular oscillator, to elevate circadian amplitude and improve energy homeostasis in metabolic disease mice. Importantly, in naturally aged mice, we recently showed that the NOB-ROR axis strengthens metabolic homeostasis and promotes energy expenditure in part via mitochondrial activation in skeletal muscle, ultimately bolstering healthy aging and survival. Therefore, NOB (a dietary flavonoid) and TRF differentially modify circadian core oscillator and feeding rhythms, performing overlapping functions to improve fitness during aging. In this proposal, we hypothesize that NOB and TRF synergistically enhance core oscillators and output rhythms to maintain a robust clock and promote healthy aging, via mechanisms impinging on molecular oscillators and mitochondrial function. In Aim 1, building on our strong preliminary studies, we will determine roles of ad libitum NOB treatment in single-cell oscillators and transcriptional/epigenetic regulation to enhance circadian amplitude in aged mice. In Aim 2, we will determine function and mechanism of an integrated NOB.TRF regiment to coordinately activate circadian rhythms and physiology in aged mice in a concerted manner. In Aim 3, we will delineate the circadian mechanisms via which NOB and TRF regulate mitochondrial respiration. Together, the proposed studies will provide key mechanistic insights into the role of circadian rhythms during aging, and pinpoint an integrated dietary regimen as a novel strategy to activate clocks and extend healthspan. The innovations include a dual-component dietary regimen coordinately enhance clock amplitude for healthy aging and a novel circa...