Metabolic assessment of iPSC-derived neurons under ketone-enriched condition: Ketone sensor development and BHB-driven metabolic adaptation

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Neurons depend on glucose to sustain their high energetic demands; yet, ketone bodies can serve as alternative substrates during ketogenic states. Here, we examined how β-hydroxybutyrate reshapes metabolism and function in human iPSC-derived neurons. Neurons generated from neuroepithelial stem cells were cultured in glucose-rich media or low-glucose media supplemented with β-hydroxybutyrate. We developed an electrochemical biosensor for ketone detection and validated its performance by cyclic voltammetry and amperometry, achieving linear sensitivity in the 0.01 to 0.1 mM range. Metabolic changes for neurons were assessed through glucose consumption and lactate production, and transcriptional profiling revealed reduced expression of selected metabolic and ketone-associated genes under ketone supplementation. Calcium imaging further showed lower firing rates in ketone exposed neurons compared with glucose conditions. Together, these results demonstrate how alternative energy substrates modulate neuronal metabolism and excitability, providing a framework to evaluate metabolic interventions for neurological disorders.