Lipophagy, a selective form of autophagy, is critical for maintaining cellular lipid homeostasis. However, understanding its dynamic regulation and pathophysiological significance in vivo has been hindered by a lack of sensitive and versatile monitoring tools. To address this gap, we generated the tfLiveDrop (mCherry-eGFP-LiveDrop) reporter mouse by integrating a tandem mCherry-eGFP fluorescent probe with the lipid droplet-targeting domain of glycerol-3-phosphate acyltransferase 4 (GPAT4, the rate-limiting enzyme in triacylglycerol synthesis), termed the LiveDrop domain. This model enables real-time, spatiotemporal visualization of lipophagic flux at single-cell resolution in living animals. We initially validated the sensitivity and specificity of the tfLiveDrop reporter in primary renal tubular epithelial cells (TECs). Systemic mapping of lipophagic activity across organs revealed pronounced heterogeneity in basal lipophagic activity under physiological conditions. Furthermore, in a model of Type 2 diabetes, we demonstrated that lipophagic flux is dysregulated in a tissue-specific manner in male mice, underscoring its pivotal role in disease-associated lipid metabolism. Notably, longitudinal tracking during kidney development uncovered a programmed wave of lipophagic activity that is essential for lipid homeostasis during renal maturation. Our findings provide a powerful and versatile platform for in vivo lipophagy research, establishing a foundation for elucidating its functional contributions to metabolic disorders and organ development.
