Abbreviations: Dilps: Drosophila insulin-like peptides IPCs: Insulin-producing cells IIS: Insulin/Insulin-like growth factor signalling PG: Prothoracic Gland 20E: 20-hydroxyecdysone L1-L3: First, Second, Third Larval Instar FB: Fat Body RNAi: RNA interference qPCR: quantitative Polymerase Chain Reaction h AEL: hours after egg laying h AL3E: hours after third instar ecdysis 2 Abstract: Growth and maturation are coordinated processes in all animals. Integration of internal cues, such as signalling pathways, with external cues such as nutritional status is paramount for an orderly progression of development in function of growth. In Drosophila, this coordination involves insulin and steroid signalling, but the mechanisms by which this occurs and how they are coordinated are incompletely understood. We show that production of the bioactive 20-hydroxyecdysone by the enzyme Shade in the fat body is a nutrientdependent process. We demonstrate that during fed conditions, Shade plays a role in growth regulation, as knockdown of shade in the fat body resulted in growth defects and perturbed expression and release of the Drosophila insulin-like peptides from the insulinproducing cells (IPCs). We identify the trachea and IPCs as direct targets through which 20hydroxyecdysone regulates insulin-signaling. The identification of the trachea-dependent regulation of insulin-signaling exposes an important variable that may have been overlooked in other studies focusing on insulin-signaling in Drosophila. Finally, we show with IPCspecific manipulations that 20E may both be a growth-promoting and growth-inhibiting signal in the IPCs acting through different nuclear receptors. Our findings provide a potentially conserved, novel mechanism by which nutrition can modulate steroid hormone bioactivation, reveal an important caveat of a commonly used transgenic tool to study IPC function and yield further insights as to how steroid and insulin signalling are coordinated during development to regulate growth and developmental timing.3