The Effect of Ketone Body Supplement β-hydroxybutyrate on Autophagy Gene Expression in Drosophila melanogaster Larval Fat Body

Abstract

Autophagy is a conserved catabolic process in eukaryotic cells in response to stress conditions such as starvation. It also plays a crucial role in maintaining cellular health by clearing damaged organelles, aggregated proteins, and intracellular pathogens. During autophagy, autophagosome vacuoles form and capture cytoplasm contents, which then fuse with lysosomes to further degrade the contents (McPhee & Baehrecke, 2009). In response to nutrient deprivation, autophagy is the key mechanism for cells to break down proteins, carbohydrates, and lipids into metabolites like amino acids, glucose, and fatty acids. These metabolites can then be used in biosynthesis pathways or further catabolized to produce ATP (He, 2022).

Autophagy can be induced under several different conditions, such as starvation with low amino acid and glucose levels. One of the main nutrient-sensing pathways that regulates autophagy is the mammalian target of rapamycin (mTOR) pathway. When it’s activated, mTOR directly inhibits autophagy by phosphorylation of Atg1 from the Atg1 complex, which is responsible for the induction of autophagosome formation (Chang & Neufeld, 2010). After the induction, the nucleation of the autophagosome membrane requires the Vps34 complex containing the core protein Atg6/Beclin 1. Other key regulators of autophagy include AMP-activated protein kinase (AMPK) and PI3K/AKT in insulin signaling.

Beta-hydroxybutyrate (BHB) is a ketone body produced by cells in response to the ketogenic diet and caloric restriction. It serves as an alternative energy source to glucose. BHB supplementation is a potential substitute for the ketogenic diet, providing similar health benefits without the need of strict dietary restriction. Previous work in our group measuring Atg8a-II/Atg8a-I ratio by western blot has found that autophagy levels in Drosophila melanogaster 3rd instar larvae fed a BHB-supplemented diet are higher than those of 3rd instar larvae fed a control diet (Becher et al., 2024). Therefore, my research aims to answer the question: how does BHB drive autophagy, a process that could conflict with the nutrient storage demand of fat body cells, at such a critical stage in Drosophila development? It’s not well understood whether BHB regulates autophagy through transcription, translation, or post-translational modifications (PTM) of key signaling proteins. My qPCR results showed that BHB supplementation does not significantly affect the transcriptional levels of Atg1, Atg6, and Atg8a. Future studies could start with investigating whether it regulates autophagy through the phosphorylation of AKT using a Western Blot approach.