Minoru Akiyama’s research while affiliated with Honda Research Institute Japan Co., Ltd. and other places

Background Hypoxia can occur in daily life, and exercise is a potential trigger. Intense exercise increases oxygen demand in muscles, potentially compromising performance by reducing ATP resynthesis. Kaempferol (KMP) increases intracellular ATP resynthesis in hypoxic cellular models. Aims We investigated the effects of KMP on oxygen utilization during exercise in a general human population. Methods Study 1 (Ramp-incremental cycling test): Overall, 20 men were randomized to four groups and administrated placebo, 2.5, 10, or 25 mg KMP for 8 days in a crossover design. Measurements of oxygen utilization efficiency slope (OUES), maximal oxygen uptake (VO2max), peak heart rate (HR), VO2 and work rate at gas-exchange threshold, minute ventilation/carbon dioxide output ratio (VE/VCO2) slope, and post-exercise muscle tightness and breathing difficulty were performed on days 1 and 8. Study 2 (Constant-load cycling test): Overall, 25 men were randomized to groups that received either a single dose of 10 mg KMP or a placebo in a crossover design. VO2/VE, VO2max, VO2, respiratory rate, HR, respiratory exchange ratio, and VE/VCO2 were measured at exercise intensities of 25%, 50%, and 75% of VO2max. Results Study 1: Both OUES and VO2max showed significant dose-dependent increase on day 1. The optimal dose and frequency for the ergogenic effects of KMP was found to be 10 mg in a single intake. Study 2: KMP improved VO2/VE and enhanced respiratory and cardiovascular indices. Conclusions KMP enhances the maximum capacity for oxygen utilization and improves oxygen utilization efficiency at several exercise levels. Trial registration Study 1: UMIN000049587 (November 23, 2022); Study 2: UMIN000049590 (November 23, 2022) (UMIN Clinical Trials Registry).

Plants exposed to hypoxic conditions have been suggested to produce more biologically active phytochemicals than those exposed to normal oxygen levels. Previously, we investigated 314 highland crop species and showed that the flavonoid kaempferol extracted from a highland quinoa grain markedly increased mitochondrial metabolism and ATP production in a hypoxic environment in vitro. Thus, we hypothesized that kaempferol would be effective during exercise under harsh conditions, in which anaerobic metabolism occurs. This study adopted a double‐blind, placebo‐controlled, crossover design to investigate the effect of a single oral dose of kaempferol (10 mg) on the athletic performance‐related indicators of 13 male university athletes (20.8 ± 0.7 years) who performed two consecutive 400‐m runs with the shortest 90‐min interval assuming qualifying and main races. Although no significant differences were observed in the 400‐m race times between the placebo and kaempferol groups, kaempferol intake markedly reduced the respiratory and heart rates during the second run (p < 0.05). In addition, kaempferol intake reduced the levels of muscle damage markers, myoglobin, and aspartate transaminase (p < 0.05). A single oral dose of kaempferol reduced the cardiopulmonary burden and muscle damage in individuals participating in 400‐m runs. Kaempferol may be a useful supplement for relieving the physical load, particularly in individuals performing strenuous exercises with high oxygen demand. Trial Registration: UMIN Clinical Trials Registry in Japan: UMIN000049588

Kaempferol (KMP) is an important flavonoid in many fruits and vegetables. Preclinical studies on KMP have reported its pharmacological effects, including antimicrobial, antioxidant, anti‐inflammatory, antitumor, antidiabetic, myocardial protective, and neuroprotective effects. Additionally, some epidemiological studies have revealed a negative association between the consumption of KMP‐containing foods and the risk of developing several disorders, such as cancer and cardiovascular diseases. Thus, although a large body of literature has demonstrated the benefits of KMP supplementation, there are no reports of clinical trials evaluating the safety of KMP aglycone administration or KMP aglycone‐rich food consumption. The purpose of this study was to evaluate the safety of a high dose of KMP aglycone by administrating KMP aglycone‐containing supplements to healthy adults. This study had a randomized, double‐blind, placebo‐controlled design and a 4‐week duration. Participants were randomly allocated to the KMP ( n = 24) or placebo ( n = 24) group. For 4 weeks, the KMP group received a capsule containing 50‐mg KMP daily, a dose approximately five times higher than the estimated human dietary intake. The placebo group received a capsule containing cornstarch‐based powder daily. The general toxicity parameters were evaluated by examining the characteristics of the participants, hematological and blood biochemical parameters, general urinalysis, qualitative urine tests, and adverse events. No clinical changes were observed in anthropometric and blood pressure measurements or blood and urine parameters in the KMP group compared to those in the placebo group. Furthermore, no adverse events owing to KMP aglycone administration occurred. The study results revealed that the consumption of 50‐mg KMP aglycone daily for 4 weeks is safe in healthy adults.

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.

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 C2C12 myotubes, to more accurately determine their physiological activities. We found intracellular ATP content in C2C12 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.