Combination of tea catechins and ornithine effectively activates the urea cycle: an in vitro and human pilot study.

PURPOSE: Accumulation of ammonia causes central and peripheral fatigue. This study aimed to investigate the synergistic effect of tea catechins and low-dose ornithine in activating the urea cycle to reduce blood ammonia levels during exercise. METHODS: We used hepatocyte-like cells derived from human-induced pluripotent stem (iPS) cells to assess the effect of tea catechins combined with ornithine on urea cycle activity. The urea production and expression of key genes involved in the metabolism of urea were investigated. We then examined the synergistic improvement in ammonia metabolism by tea catechins in combination with ornithine in a human pilot study. RESULTS: Tea catechins combined with ornithine increased urea cycle activity in hepatocyte-like cells derived from human iPS cells. Intake of 538.6 mg of tea catechins with 1592 mg of ornithine for 2 consecutive days during exercise loading suppressed the exercise-induced increase in the blood ammonia concentration as well as stabilized blood glucose levels. CONCLUSION: Controlling the levels of ammonia, a toxic waste produced in the body, is important in a variety of situations, including exercise. The present study suggests that a heterogeneous combination of polyphenols and amino acids efficiently suppresses elevated ammonia during exercise in humans by a mechanism that includes urea cycle activation. TRIAL REGISTRATION: This study was registered in the University Hospital Medical Information Network Clinical Trial Registry (No. UMIN000035484, dated January 8, 2019).

DOK7 Gene Therapy Enhances Neuromuscular Junction Innervation and Motor Function in Aged Mice.

Muscle denervation at the neuromuscular junction (NMJ), the essential synapse between motor neuron and skeletal muscle, is associated with age-related motor impairment. Therefore, improving muscle innervation at aged NMJs may be an effective therapeutic strategy for treating the impairment. We previously demonstrated that the muscle protein Dok-7 plays an essential role in NMJ formation, and, indeed, its forced expression in muscle enlarges NMJs. Moreover, therapeutic administration of an adeno-associated virus vector encoding human Dok-7 (DOK7 gene therapy) suppressed muscle denervation and enhanced motor activity in a mouse model of amyotrophic lateral sclerosis (ALS). Here, we show that DOK7 gene therapy significantly enhances motor function and muscle strength together with NMJ innervation in aged mice. Furthermore, the treated mice showed greatly increased compound muscle action potential (CMAP) amplitudes compared with the controls, suggesting enhanced neuromuscular transmission. Thus, therapies aimed at enhancing NMJ innervation have potential for treating age-related motor impairment.

TRPM8-mediated cutaneous stimulation modulates motor neuron activity during treadmill stepping in mice.

Motor units are generally recruited from the smallest to the largest following the size principle, while cutaneous stimulation has the potential to affect spinal motor control. We aimed to examine the effects of stimulating transient receptor potential channel sub-family M8 (TRPM8) combined with exercise on the modulation of spinal motor neuron (MN) excitability. Mice were topically administrated 1.5% icilin on the hindlimbs, followed by treadmill stepping. Spinal cord sections were immunostained with antibodies against c-fos and choline acetyltransferase. Icilin stimulation did not change the number of c-fos+ MNs, but increased the average soma size of the c-fos+ MNs during low-speed treadmill stepping. Furthermore, icilin stimulation combined with stepping increased c-fos+ cholinergic interneurons near the central canal, which are thought to modulate MN excitability. These findings suggest that TRPM8-mediated cutaneous stimulation with low-load exercise promotes preferential recruitment of large MNs and is potentially useful as a new training method for rehabilitation.

Ginger extract prevents high-fat diet-induced obesity in mice via activation of the peroxisome proliferator-activated receptor δ pathway.

The initiation of obesity entails an imbalance wherein energy intake exceeds expenditure. Obesity is increasing in prevalence and is now a worldwide health problem. Food-derived peroxisome proliferator-activated receptor δ (PPARδ) stimulators represent potential treatment options for obesity. Ginger (Zingiber officinale Roscoe) was previously shown to regulate the PPARγ signaling pathway in adipocytes. In this study, we investigated the antiobesity effects of ginger in vivo and the mechanism of action in vitro. Energy expenditure was increased, and diet-induced obesity was attenuated in C57BL/6J mice treated with dietary ginger extract (GE). GE also increased the number of Type I muscle fibers, improved running endurance capacity and upregulated PPARδ-targeted gene expression in skeletal muscle and the liver. 6-Shogaol and 6-gingerol acted as specific PPARδ ligands and stimulated PPARδ-dependent gene expression in cultured human skeletal muscle myotubes. An analysis of cellular respiration revealed that pretreating cultured skeletal muscle myotubes with GE increased palmitate-induced oxygen consumption rate, which suggested an increase in cellular fatty acid catabolism. These results demonstrated that sustained activation of the PPARδ pathway with GE attenuated diet-induced obesity and improved exercise endurance capacity by increasing skeletal muscle fat catabolism. 6-Shogaol and 6-gingerol may be responsible for the regulatory effects of dietary ginger on PPARδ signaling.

Menaquinone-4 enhances testosterone production in rats and testis-derived tumor cells.

BACKGROUND: Vitamin K is essential for the posttranslational modification of various Gla proteins. Although it is widespread in several organs, including the testis, the function of vitamin K in these organs is not well characterized. In this study, we investigated the function of vitamin K in the testis and analyzed its role in steroidogenesis. METHODS: Eight-week-old male Wistar rats were fed a diet supplemented with menaquinone-4 (MK-4, 75 mg/kg diet), one of the predominant K2 vitamins present in the testis, for 5 weeks. In vivo testosterone levels of the rats' plasma and testes were measured by enzyme-linked immunosorbent assay, and in vitro testosterone levels of testis-derived tumor cells (I-10 cells) maintained in Ham's F-10 medium with 10% fetal bovine serum were measured following treatment with MK-4 (0 to 100 µM) at several time points. Testosterone and cellular protein levels were analyzed with respect to their effects on steroidogenesis. RESULTS: Testosterone levels in the plasma and testes of MK-4-fed rats were significantly increased compared to those of control rats, with no obvious differences in plasma luteinizing hormone levels. Secreted testosterone levels from I-10 cells were elevated by MK-4, but not by vitamin K1, in a dose-dependent manner independent of cAMP treatment. Western blot analysis revealed that expression of CYP11A, the rate-limiting enzyme in steroidogenesis, and phosphorylation levels of protein kinase A (PKA) and the cAMP response element-binding protein were all stimulated by the presence of MK-4. Enhancement of testosterone production was inhibited by H89, a specific inhibitor of PKA, but not by warfarin, an inhibitor of γ-glutamylcarboxylation. CONCLUSIONS: MK-4 stimulates testosterone production in rats and testis-derived tumor cells via activation of PKA. MK-4 may be involved in steroidogenesis in the testis, and its supplementation could reverse the downregulation of testosterone production in elders.

Coffee polyphenols suppress diet-induced body fat accumulation by downregulating SREBP-1c and related molecules in C57BL/6J mice.

The prevalence of obesity is increasing globally, and obesity is a major risk factor for type 2 diabetes and cardiovascular disease. We investigated the effects of coffee polyphenols (CPP), which are abundant in coffee and consumed worldwide, on diet-induced body fat accumulation. C57BL/6J mice were fed either a control diet, a high-fat diet, or a high-fat diet supplemented with 0.5 to 1.0% CPP for 2-15 wk. Supplementation with CPP significantly reduced body weight gain, abdominal and liver fat accumulation, and infiltration of macrophages into adipose tissues. Energy expenditure evaluated by indirect calorimetry was significantly increased in CPP-fed mice. The mRNA levels of sterol regulatory element-binding protein (SREBP)-1c, acetyl-CoA carboxylase-1 and -2, stearoyl-CoA desaturase-1, and pyruvate dehydrogenase kinase-4 in the liver were significantly lower in CPP-fed mice than in high-fat control mice. Similarly, CPP suppressed the expression of these molecules in Hepa 1-6 cells, concomitant with an increase in microRNA-122. Structure-activity relationship studies of nine quinic acid derivatives isolated from CPP in Hepa 1-6 cells suggested that mono- or di-caffeoyl quinic acids (CQA) are active substances in the beneficial effects of CPP. Furthermore, CPP and 5-CQA decreased the nuclear active form of SREBP-1, acetyl-CoA carboxylase activity, and cellular malonyl-CoA levels. These findings indicate that CPP enhances energy metabolism and reduces lipogenesis by downregulating SREBP-1c and related molecules, which leads to the suppression of body fat accumulation.

Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet-induced obesity by activating AMPK.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is implicated in the control of energy metabolism and is considered to be a molecular target for the suppression of obesity and the treatment of metabolic syndrome. Here, we identified and characterized nootkatone, a constituent of grapefruit, as a naturally occurring AMPK activator. Nootkatone induced an increase in AMPKalpha1 and -alpha2 activity along with an increase in the AMP/ATP ratio and an increase the phosphorylation of AMPKalpha and the downstream target acetyl-CoA carboxylase (ACC), in C(2)C(12) cells. Nootkatone-induced activation of AMPK was possibly mediated both by LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinase. Nootkatone also upregulated PPARgamma coactivator-1alpha in C(2)C(12) cells and C57BL/6J mouse muscle. In addition, administration of nootkatone (200 mg/kg body wt) significantly enhanced AMPK activity, accompanied by LKB1, AMPK, and ACC phosphorylation in the liver and muscle of mice. Whole body energy expenditure evaluated by indirect calorimetry was also increased by nootkatone administration. Long-term intake of diets containing 0.1% to 0.3% (wt/wt) nootkatone significantly reduced high-fat and high-sucrose diet-induced body weight gain, abdominal fat accumulation, and the development of hyperglycemia, hyperinsulinemia, and hyperleptinemia in C57BL/6J mice. Furthermore, endurance capacity, evaluated as swimming time to exhaustion in BALB/c mice, was 21% longer in mice fed 0.2% nootkatone than in control mice. These findings indicate that long-term intake of nootkatone is beneficial toward preventing obesity and improving physical performance and that these effects are due, at least in part, to enhanced energy metabolism through AMPK activation in skeletal muscle and liver.