Resveratrol mainly stimulates the glycolytic ATP synthesis flux and not the mitochondrial one: A saturation transfer NMR study in perfused and isolated rat liver

Abstract Our aim was to monitor the effects of resveratrol (RSV) on the respective contribution of glycolysis and oxidative phosphorylation on the unidirectional flux of ATP synthesis in whole isolated rat liver perfused with Krebs-Henseleit Buffer (KHB). The rate of tissular ATP supply was measured directly by monitoring the chemical exchange Pi toward ATP with saturation transfer (ST) 31 P nuclear magnetic resonance, a method applied for the first time for studying the effects of RSV. ST allows the measurement of the total cellular Pi → ATP chemical exchange; after specific inhibition of glycolysis with iodacetate, ST could provide the Pi → ATP flux issued from mitochondria. This latter was compared to mitochondrial ATP turn-over evaluated after chemical ischemia (CI), performed with specific inhibition (KCN) of oxidative phosphorylation, and measured by standard 31 P NMR spectroscopy. In controls (KHB alone), the apparent time constant ( k s ) of Pi exchange toward ATP as measured by ST was 0.48 ± 0.04 s −1 leading to a total ATP synthesis rate of 37 ± 3.9 μmol min −1  g −1 . KHB + RSV perfusion increased k s (+52%; p  = 0.0009 vs. KHB) leading to an enhanced rate of total ATP synthesis (+52%; p  = 0.01 vs. KHB). When glycolysis was previously inhibited in KHB, both k s and ATP synthesis flux dramatically decreased (−87% and −86%, respectively, p vs. KHB without inhibition), evidencing a collapse of Pi-to-ATP exchange. However, glycolysis inhibition in KHB + RSV reduced to less extent k s (−41%, p  = 0.0005 vs. KHB + RSV without inhibition) and ATP synthesis flux (−18%). Using the CI method in KHB and KHB + RSV, KCN addition after glycolysis inhibition induced a rapid fall to zero of the ATP content. The mitochondrial ATP turnover R ( t 0) and its time constant k d mito were similar in KHB (1.18 ± 0.19 μmol min −1  g −1 and 0.91 ± 0.13 min −1 ) and KHB + RSV (1.36 ± 0.26 μmol min −1  g −1 and 0.77 ± 0.18 min −1 ). Since mitochondrial ATP turnover was not increased by RSV, the stimulation of Pi-to-ATP exchange by RSV mainly reflected an increase in glycolytic ATP synthesis flux. Moreover, the maintenance by RSV of a high level of Pi-to-ATP exchange after glycolysis inhibition evidenced a protective effect of the polyphenol, in agreement with our previous hypothesis of a stimulation of substrate flux throughout the glycolysis 3-carbon step.