Description: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 observed strong correlation between daily daily rhythms of protein O- linked N-acetylglucosaminylation (O-GlcNAcylation) 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, which could disrupt circadian health. We performed targeted metabolomic analysis on hexosamine biosynthetic pathway (HBP), which produces UDP-GlcNAc (the substrate for O-GlcNAcylation), to evaluate the daily activity of HBP enzymes under TRF conditions. We found glutamine--fructose-6-phosphate amidotransferase (GFAT) mediates this buffering mechanism.