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Kaempferol (KMP) has numerous important biological functions, and we recently showed that it remarkably increased intracellular adenosine triphosphate (ATP) content in C2C12 myotubes under hypoxic conditions. Since intracellular ATP is generated by aerobic energy metabolism or anaerobic glycolysis, hypoxia inducible factor-1α (HIF-1α) has been shown to be associated with metabolic remodeling and causes metabolic shift from aerobic energy metabolism to anaerobic glycolysis in response to hypoxic conditions. Here, we investigate the effects of KMP under hypoxic conditions on the stabilization of HIF-1α in C2C12 myotubes and its underlying molecular mechanisms. Constitutive HIF-1α protein expression was observed in C2C12 myotubes, and its expression under hypoxic conditions was remarkably suppressed by KMP by reducing its stability; thus, resulting in an increase in ATP content. Furthermore, KMP strikingly increased the ubiquitination of HIF-1α and promoted its degradation via the ubiquitin proteasome system. Inhibition of HIF-1α by KMP resulted in the abrogation of the expression of glycolytic enzymes such as lactate dehydrogenase A and pyruvate dehydrogenase kinase isozyme 1. In addition, the metabolome profiling showed that KMP promoted oxidative energy production, while the mitochondrial complex activity assay indicated that KMP increased the activity of mitochondrial complex IV. Finally, we showed that KMP inhibited HIF-1α expression and increased intracellular ATP content in the soleus muscle of rats. Taken together, these results suggest that KMP increases intracellular ATP content under hypoxic conditions by suppressing the HIF-1α stabilization and/or by enhancing the mitochondrial complex IV activity in muscle. [ABSTRACT FROM AUTHOR]

[Display omitted] • ATP increasing effect of 65 phytochemicals was evaluated under hypoxic conditions. • Kaempferol increased intracellular ATP content under hypoxic conditions. • Required structure in flavonoids to increase intracellular ATP content was clarified. • Hypoxic environment increased biosynthesis of kaempferol in crops. Hypoxic conditions in the body have been reported to occur in aging and exercising muscles. It has been suggested that a reduction in ATP, due to hypoxia, may contribute to related declines in muscle performance. In this study, we established hypoxic assay systems and evaluated the effects of 65 phytochemicals on intracellular ATP content in C 2 C 12 myotubes, to more accurately determine their physiological activities. We found intracellular ATP content in C 2 C 12 myotubes to be reduced, under hypoxic conditions. However, kaempferol markedly increased it in this assay systems, by activating oxidative metabolism. The level of kaempferol in crops cultivated in highland areas or an artificial hypoxic environment, was significantly increased, while that of quercetin was either unchanged or significantly decreased. These results suggest crops may biosynthesize kaempferol to adapt to hypoxic environments, and that highland residents may benefit from eating such crops, with adaptive consequences. [ABSTRACT FROM AUTHOR]