Substrate‐specific mitochondrial dysfunction and metabolomic profiles in type 2 diabetic rat hearts

Abstract Type 2 diabetes (T2D) greatly alters cardiac fuel handling, yet how mitochondrial function adapts to the diabetic substrate environment remains unclear. This study investigated substrate‐specific cardiac mitochondrial bioenergetics from a T2D rat model induced by a high‐fat diet and low‐dose streptozotocin. High‐resolution respirometry and fluorimetry were used to measure mitochondrial O 2 flux, ATP flux, reactive oxygen species production rate and mitochondrial membrane potential in cardiac tissue homogenates in two key substrate conditions: carbohydrate and fatty acid. Liquid chromatography–mass spectrometry was used to analyse the abundance of metabolites involved in the Krebs cycle and in various fatty acid metabolic pathways. Carbohydrate‐supported mitochondrial respiration, ATP flux, reactive oxygen species production and mitochondrial membrane potential were preserved in T2D myocardium. In contrast, fatty acid‐supported mitochondrial respiration and ATP flux in oxidative phosphorylation were significantly decreased despite increased myocardial abundance of several fatty acid species, including palmitoleic acid, cis ‐8‐heptadecenoic acid and linoleic acid. Metabolite intermediates of the Krebs cycle were largely unchanged. These findings reveal a substrate‐specific energetic defect in the diabetic heart, in which excess fatty acid supply is not matched by mitochondrial oxidative capacity, leading to metabolic inflexibility and impaired ATP generation. This work provides mechanistic insight into how nutrient overload contributes to mitochondrial inefficiency in T2D and establishes a foundation for future studies targeting lipid–mitochondria interactions.