In the cardiac context, glucose and glycolysis play pivotal roles in supporting anaplerosis and potentially influencing the oxidation of d-β-hydroxybutyrate (βHB). The presence of glycogen, serving as a reservoir for glucose, could also contribute to anaplerosis. To delve into the intricate connections between glycogen content, βHB oxidation, glycolytic rates, and their consequential impact on energy dynamics, an isolated rat heart model was employed. Hearts with high glycogen (HG) and low glycogen (LG) levels were perfused with 11 mM [5-3H] glucose and/or 4 mM [14C] βHB to assess glycolysis and βHB oxidation, respectively. Subsequently, freeze-clamping was carried out for glycogen and metabolomic analyses. The ratio of free cytosolic [NAD+]/[NADH] and mitochondrial [Q+]/[QH2] was estimated using the lactate dehydrogenase and succinate dehydrogenase reactions. 31P-nuclear magnetic resonance spectroscopy was utilized to measure phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations. Notably, βHB oxidation rates in LG hearts were found to be half of those in HG hearts, exhibiting a direct correlation with glycogen content. Remarkably, βHB oxidation led to a reduction in glycolysis across all heart conditions. In glycogen-rich hearts perfused solely with βHB, glycogenolysis was twofold compared to hearts perfused with both βHB and glucose. This latter group demonstrated elevated levels of glycolytic intermediates, specifically fructose 1,6-bisphosphate and 3-phosphoglycerate, alongside a higher free cytosolic [NAD+]/[NADH] ratio. The influence of βHB oxidation was further evident through heightened levels of Krebs cycle intermediates, such as citrate, 2-oxoglutarate, and succinate. Additionally, the total NADP/H pool increased, mitochondrial [Q+]/[QH2] decreased, and the calculated free energy of ATP hydrolysis (ΔGATP) was elevated. Intriguingly, while βHB oxidation exerted an inhibitory effect on glycolysis, the reserves of glycolytic intermediates remained intact, and cytosolic free NAD sustained its oxidized state. Furthermore, βHB oxidation in isolation not only amplified Krebs cycle intermediates but also resulted in reduced mitochondrial Q levels and an enhanced ΔGATP. In summation, our findings underscore the facilitating role of glycogen in promoting cardiac βHB oxidation through anaplerosis.
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