Mitochondrial homeostasis of endocrine organ navigates nutrient restriction checkpoint

Steroid hormones are powerful endocrine regulators, but little is known about the integration of their production with environmental conditions. Here, we use Drosophila prothoracic gland (PG) to examine how insect steroid hormone production navigates a developmental checkpoint critical for juvenile-to-adult transition. Extensive transcriptome analysis of the PGs revealed that starvation during pre-nutrient restriction checkpoint (NRC) downregulated substantial mitochondria-related genes. We then discovered that pre-NRC starvation reduced prothoraciotropic hormone signaling, insulin signaling, and TORC1 activity in PG cells, which prevented mitochondrial fragmentation and import of Disembodied, a key steroidogenic enzyme. Ultimately, this starvation caused severe mitophagy and proteasome dysfunction, blocking ecdysteroidgenesis and metamorphosis. By contrast, post-NRC starvation did not impair mitochondrial homeostasis in PG cells, but reduced Sit expression and induced moderate autophagy to promote ecdysteroidgenesis, leading to precocious metamorphosis. This study constitutes a paradigm for exploring how steroid hormones themselves are controlled in response to environmental stress like starvation. Overall design: The RGs of control or starved w1118 larvae were dissected in PBS in batches of 10–40 at a time, and pooled into three biological replicates for every treatment. About 1,200 RGs were pooled per sample, which yielded ~1-5 µg of RNA required for sequencing. Total RNA was extracted using the miRNeasy Micro Kit (QIAGEN, Germany), then stored at ~80 °C. RNA quality, especially DNA contamination, was monitored on 1.5% agarose gels. RNA concentration was measured using NanoDrop 2000 (Thermo Fisher Scientific, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). High-quality RNA samples from Drosophila RGs were sent to Biomarker Technologies Corporation (Beijing, China) for construction of cDNA libraries and sequencing.