E abdomen of PLD Inhibitor drug TKgNPFRNAi animals (Fig. 2f). Notably, upregulation of Acetyl-CoA carboxylase (ACC) was not reproduced with qPCR (Fig. 2f). These information recommend that TKgNPFRNAi animals are inside the starved-like status regardless of taking in extra meals, and that haemolymph glucose levels cannot be maintained even with the activation of gluconeogenesis and lipolysis in TKgNPFRNAi animals. We hypothesise that, owing to the starved-like status,the loss of midgut NPF function could possibly result in an abnormal consumption of TAG, resulting in the lean phenotype. Midgut NPF responds to dietary sugar. SIRT1 Modulator Purity & Documentation Considering that EECs can sense dietary nutrients, we surmised that dietary nutrients impact NPF production and/or secretion in midgut EECs. We thus compared NPF protein and mRNA levels in flies fed typical food or starved for 48 h with 1 agar. Following 48 h of starvation, NPF protein in midgut EECs was significantly elevated (Fig. 3a, b), though its transcript in the intestine was reduced (Fig. 3c). These information recommend that the increased accumulation of NPF protein in EECs upon starvation just isn’t resulting from upregulation of NPF mRNA expression level, but rather due to posttranscriptional regulation. This predicament was very related for the case of mating-dependent alter of NPF protein level, and may reflect the secretion of NPF protein from EECs17. Considering that the higher accumulation of NPF protein without having NPF mRNA improve indicate a failure of NPF secretion, we hypothesised that starvation suppresses NPF secretion from EECs. To recognize specific dietary nutrients that have an effect on NPF levels in EECs, immediately after starvation, we fed flies a sucrose or Bacto peptone diet regime as exclusive sources of sugar and proteins, respectively. Interestingly, by supplying sucrose, the levels of each of NPF protein and NPF mRNA inside the gut reverted to the levels equivalent to ad libitum feeding situations (Fig. 3a, b). In contrast, Bacto peptone administration didn’t reduce middle midgut NPF protein level, but rather enhanced each NPF protein and NPF mRNA levels (Fig. 3c). These information imply that midgut NPF is secreted primarily in response to dietary sugar, but not proteins. This sucrosedependent NPF secretion was observed in flies fed a sucrose medium for six h just after starvation, whereas a 1h sucrose restoration had no impact on NPF accumulation (Supplementary Fig. 6a). Sugar-responsive midgut NPF production is regulated by the sugar transporter Sut1. In mammals, the sugar-stimulated secretion of GLP-1 is partly regulated by glucose transporter two, which belongs to the low-affinity glucose transporter solute carrier family members 2 member 2 (SLC2)27,28. In D. melanogaster, a SLC2 protein, Glucose transporter 1 (Glut1), within the Burs+ EECs regulates sugar-responsible secretion and Burs mRNA expression11. Nevertheless, knockdown of Glut1 did not affect NPF mRNA nor NPF protein abundance in EECs (Supplementary Fig. 6b, c). As a result, we next examined which SLC2 protein, aside from Glut1, regulates NPF levels in the gut. You’ll find over 30 putative homologues of SLC2 in the D. melanogaster genome29. Of those, we focused on sugar transporter1 (sut1), because its expression has been described inside the intestinal EECs by FlyGut-seq project30 and Flygut EEs single-cell RNA-seq project31. To confirm sut1 expression, we generated a sut1Knock-in(KI)-T2A-GAL4 strain utilizing CRISPR/Cas9-mediated homologous recombination32,33. Consistent with these transcriptomic analyses, sut1KI-T2A-GAL4 expression was observed inside the EECs, including NPF+ EECsNATURE COMM.