Fig. 3: Dietary L-Glu inhibits NPF secretion from EECs.

a, b Representative images a and quantification b of NPF staining after ingestion of different foods. n = 75 in each group. c Quantification of NPF staining after feeding of single L-amino acids. Red dash line boxes indicate the two AAs, L-Asn and L-Glu, that significantly elevated NPF intensity. n = 75 in each group. d Representative images of NPF staining after feeding of 5% sucrose, 5% sucrose +1% L-Glu or 5% sucrose +1% L-Asn. e Normalized NPF mRNA levels after feeding different food by RT-qPCR. Each genotype corresponded to 3 biological replicates of 50 midguts each. f, g Representative images f and quantification g of pANF-EMD staining (green) after ingestion of different food. n = 30 in each group. h, i Under 1% L-Glu feeding condition, representative images h and quantification i of NPF staining in EECs of control and tap^1.3-B-Gal4>TrpA1 flies at 18 °C and 30 °C. n = 75 in each group. j Food intake of control and tap^1.3-B-Gal4>TrpA1 flies under 1% L-Glu feeding condition at 18 °C and 30 °C. k, l High-sugar (SCD + 10% sucrose), high-fat (SCD + 25% coconut oil) and high-protein (SCD + 10%yeast) food consumption of control and tap^1.3-B-Gal4 > NPF^RNAi flies k or esg^P>scute^RNAi flies at 3 d AE l measured using the dye-based food intake. m Schematic representation of the regulation of feeding by L-Glu that acts not only via neural perception, but also promotes appetite by inhibiting NPF release. n Food intake of control and tap^1.3-B-Gal4 > NPF^RNAi flies under different combinations of treatment (400 mM sucrose, 1% L-Glu, and NPF injection). Data are represented as mean ± SD. Significance was determined using two-sided unpaired t-test b, c, e, g, i–l, n. n, number of EECs b, c, g, i, number of groups performed for quantification of food intake (5 flies in each group) j, n, or number of groups (20 flies in each group) performed for quantification of food consumption k, l. Source data are provided as a Source Data file. Scale bars, 20 μm.