Alternative Oxidase Confers Thermogenesis and Increased Growth to Drosophila Larvae under Severe Cold Stress

Alternative oxidases (AOX) are non‐proton‐pumping enzymes present in most groups of organisms, but naturally absent in the mitochondria of vertebrates and insects. However, its xenotopic expression in mammalian and Drosophila models have suggested an immense therapeutic potential, as diverse phenotypes related to mitochondrial diseases have been attenuated. The expression of this enzyme creates an extra pathway for oxygen reduction when the cytochrome c segment of the electron transfer system is overloaded, relieving possible oxidative stress and providing beneficial effects. AOX is known to function in thermogenesis in plants due to its mitochondrial uncoupling activity, so understanding the inherent properties of AOX expressed in metazoans and how they interfere with the metabolism and physiology of model organisms under stress conditions is essential. We have been investigating the effects of temperature and have showed previously that D. melanogaster lines constitutively expressing AOX from Ciona intestinalis develop faster and have larval and pupal viability higher than that of control flies at low temperatures. Here we show that AOX‐expressing larvae are also more active and accumulate ~12,5% more body mass when cultured at the severely stressful temperature of 12°C. Using infrared thermography, we observed that these larvae lose body heat significantly less pronounced, maintaining their body temperature ~0.2°C higher than controls. To understand these effects we have analyzed larval mitochondrial respiration and show a reconfiguration of the electron transfer pathways that sustain oxygen consumption: AOX‐expressing flies have a ~30% decrease in glycerol‐3‐phosphate dehydrogenase (mGPDH)‐driven oxygen consumption, which is compensated by a ~30% increase in complex I (CI)‐driven oxygen consumption, at 12°C. AOX inhibition leads to a ~37% decrease in mGPDH‐driven respiration, suggesting a functional interaction between mGPDH and AOX. Because mGPDH is also a non‐proton‐pumping enzyme, this interaction would ultimately uncouple mitochondria. At low temperatures, this configuration becomes highly functional, especially because mGPDH appears naturally less sensitive to cold, dissipating the energy of electron transfer as heat. Assuming that the increased CI‐driven respiration may stimulate the reactions in the tricarboxylic acid cycle, we speculate the increased body mass and growth in AOX larvae may be explained by increased cataplerosis. In summary, our results suggest the increase in cold‐dependent fitness observed for AOX lines is caused by a combination of heat production via mGPDH‐linked mitochondrial uncoupling and increased CI‐linked cataplerosis.