Fig. 4. Glutamine-fructose-6-phosphate aminotransferase (GFAT) is a key enzyme that is regulated by circadian clock.

a Line graphs showing daily rhythms of HBP metabolites detected by targeted metabolomics in WT (w¹¹¹⁸) flies fed between ZT21-3 (blue) or ZT9-15 (yellow) (n = 6; p values from RAIN analysis are presented under each graph). Data were presented as mean ± SEM. b, c Expression of gfat1 and gfat2 mRNA in three groups of flies: WT RF21-3, per⁰ RF21-3, and WT RF9-15. (n = 3; gfat1 mRNA: WT RF21-3: p = 0.73, WT RF9-15: p = 0.59, per⁰ RF21-3: p = 0.76; gfat2 mRNA: WT RF21-3: p = 1.58E−05, WT RF9-15: p = 0.00019, per⁰ RF21-3: p = 3.89E−06, RAIN); (gfat2 mRNA: WT vs per⁰ RF21-3: mesor p = 0.23, amplitude p = 0.53, phase p = 0.66; WT RF21-3 vs RF9-15: mesor p = 0.95, amplitude p = 0.40, phase p = 1.12E−12; CircaCompare). d Line graph representing daily GFAT enzymatic activity in WT (w¹¹¹⁸) flies fed between ZT21-3 or ZT9-15 and per⁰ flies fed between ZT21-3 (n = 3; WT RF21-3: p = 5.73E−07, WT RF9-15: p = 0.041, per⁰ RF21-3: p = 0.92, RAIN; WT RF21-3 vs RF9-15: mesor p = 0.14, amplitude p = 0.01, phase p = 0.027, CircaCompare). b–d Data are presented as mean ± SEM. Gray shading indicates the dark period of LD cycle. Colored bars at the top of graphs denote the feeding periods for TRF treatment groups, RF21-3 (blue) and RF9-15 (yellow). The asterisks denote the comparison between WT RF21-3 and per⁰ RF21-3 using two-way ANOVA with post-hoc Tukey’s HSD tests. *p < 0.05, ****p < 0.0001.