Abstract
Mitochondria are the central hubs of energy metabolism, integrating carbohydrate, lipid, and amino acid oxidation to produce ATP through the tricarboxylic acid cycle and oxidative phosphorylation. These organelles also regulate energy homeostasis via redox signaling and substrate exchange between cellular compartments. Mitochondrial redox shuttles maintain the balance between cytosolic and mitochondrial NAD(P)H pools by transferring reducing equivalents across membranes. Among these, the glycerol-phosphate shuttle (GPSh) connects glycolysis with mitochondrial oxygen consumption, regenerating cytosolic NAD+ while transferring electrons into the electron transport system. Although GPSh ensures continuous glycolytic flux, its lower energy yield may favor heat dissipation over ATP synthesis. In the present work, we investigated how substrate utilization shapes energy coupling and thermogenesis in Drosophila melanogaster flight muscle using chip calorimetry. Calorimetric assays revealed that G3P oxidation generated significantly more heat than complex I substrates, demonstrating its high thermogenic potential. When glucose was supplied to intact flight muscles, inhibition of the GPSh with iGP1 decreased heat generation by ~ 85%, highlighting its importance for cytosolic NAD⁺ turnover and glycolytic flux. In summary, GPSh serves as a key mechanism for sustaining NAD+ regeneration in glycolysis but operates with low energy efficiency, leading to increased heat production in highly metabolically active tissues such as insect flight muscle.