Hexosamine biosynthetic pathway integrates circadian and metabolic signals to regulate daily rhythms in protein O-linked N-acetylglucosaminylation

SUMMARY The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we showed that cellular protein O-linked N-acetylglucosaminylation (O-GlcNAcylation) exhibits robust 24-hour rhythm and is a key post-translational mechanism that regulates circadian physiology. We observed strong correlation between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms were severely dampened when we subjected flies to time-restricted feeding (TRF) at unnatural feeding time. This suggests the presence of a clock-regulated buffering mechanism that prevents excessive O-GlcNAcylation at non-optimal times of the day-night cycle. We found that this buffering mechanism is mediated by glutamine-fructose-6-phosphate amidotransferase (GFAT) activity, which is regulated through integration of circadian and metabolic signals. Finally, we generated a mathematical model to describe the key factors that regulate daily O-GlcNAcylation rhythm.