21st Century COE Program Human Nutritional Science on Stress Control
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Dr. Nakaya's Labo
Prof. Yutaka Nakaya
Department of Nutrition and Metabolism
http://www.tokushima-u.ac.jp/English/englishtop.html
Dr. Nakaya's Labo
In our department, several projects are going on, including function of food, insulin resistance and it signaling pathways, clinical nutrition, and microbiology. Especially, we tried to clarify molecular mechanism of food, such as ginsenosides, which have not been studied extensively from the view point of Western science. We also tried to find new mechanism as well as new nutrients which have anti-stress action. Recently, we have established a high weel-running line of rats, named SPORTS (Spontaneously -Running -Tokushima –Shikoku) which was first discovered in an outbred strain of Wistar rats. We investigated mechanism of high weel running, and using this animal model we studied mechanism of adaptation to stress and effect of endurance exercise.
1. Functional food
a. Molecular mechanism of action of Ginsenosides
Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia as well as psychological depression. We postulate that ginsenoside Re, an active component of Panax Gingsen, enhances adaptation to stress by opening potassium channel.
Fig. 1 Activation of K channels by ginsenoside Re
We provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K+ current in cardiac muscle and large conductance Ca activated K channel in smooth muscle cells (Fig 1). The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase(PI3K), Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K+ channel activation and protection against ischemia-reperfusion injury.
Opening of K channel counteracts depolarization of the cells in cardiovascular, endocrine as well as nervous systems. Responses to mental stress involve activation of the sympathetic nervous system and secretion of epinephrine from the adrenal medulla, as well as activation of the hypothalamo-pituitary adrenal axis, the latter resulting in the secretion of glucocorticoids from the adrenal cortex. Hyperpolarization of the membrane inhibits excess responses to the stimuli in neuroendcrine system, which plays an important role in modulation of stress. In addition, hyperpolarization also act on target organ as a protective mechanism and induces less damage on the tissues.
We are also studying the effect of active metabolites of ginsenosides in the body.
(Mol Pharmacol 2006, Clin Exp Pharmacol Physiol 2001)
b. Taurine
Taurine is a normal constituent of the human diet and is ubiquitous in tissues. Taurine is a potent antioxidant and prevents tissue injury as a result of antioxidation. Most patients with diabetes have hyperinsulinemia and insulin resistance, in which the free radical oxidation of ß cells is not a cause of diabetes. In addition to antioxidant action, taurine also plays an important role in lipid metabolism, eg, such as in the enhancement of bile acid formation, which might improve lipid metabolism and insulin resistance in type 2 diabetes. Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a model of type 2 diabetes with insulin resistance. These rats also develop hyperphagia, hyperinsulinemia, obesity, and diabetes. We examined whether taurine is capable of improving insulin sensitivity and diabetic complications in OLETF rats.
Abdominal fat accumulation, hyperglycemia, and insulin resistance were significantly lower in the taurine-supplemented group than in the unsupplemented group. Serum and liver concentrations of triacylglycerol and cholesterol were significantly higher in the OLETF rats than in the control rats and were significantly lower in the taurine-supplemented group than in the unsupplemented group, presumably because of the increased secretion of cholesterol into bile acid. Taurine-supplemented rats also showed higher nitric oxide secretion, evidenced by increased urinary excretion of nitrite.
The results indicate that taurine effectively improves metabolism in OLETF rats by decreasing serum cholesterol and triacylglycerol, presumably via increased secretion of cholesterol into bile acid and decreased production of cholesterol because of increased nitric oxide production. (Am J Clin Nutr 2000; Obes Res 2004)

Taurine is reported to increase contractility of skeletal muscle and cardiac myocyte, which can increase exercise performance. Then, we aimed to clarify taurine's effect on chronic endurance exercise, especially accumulation of lactic acid (LA), a marker of fatigue and ability of aerobic exercise, and urinary secretion of 3-methylhistidine (3-MH), a marker of muscle breakdown in rats. After exercise blood levels of LA and urinary excretion of 3-MH were significantly increased and this increase was significantly less in those with chronic treatment of taurine. Taurine treatment also significantly decreased fat accumulation and blood levels of cholesterol and triglyceride, which might improve insulin resistance and utilization of fat and glucose. These results indicate taurine treatment is useful for reducing physical fatigue and muscle damage during exercise training in rats, presumably due to antioxidant property and improvement of muscle and cardiac functions by taurine. (J Nutr Sci Vitaminol 2003)
2. Clinical Nutrition
a. Liver cirrhosis and Late evening snack with BCAA mixture:
Patients with cirrhosis develop a catabolic state of starvation more rapidly than do normal subjects because of lack of store of glycogen in the liver. A late evening snack improves the catabolic state in patients with advanced liver cirrhosis. We tested whether long-term (3 mo) late evening snacking that included a branched-chain amino acid (BCAA)-enriched nutrient mixture produces a better nutritional state and better quality of life than ordinary food in patients with hepatitis C virus-positive liver cirrhosis in a multicenter, randomized study.
Serum albumin level, nitrogen balance, respiratory quotient and easy fatigability were significantly improved by the BCAA mixture but not by ordinary food, although total and late-evening energy intakes were similar in the two groups. Converstion rate of fat was significantly decreased by BCAA mixture but not in ordinary meals, suggesting that BCAA mixture could improve starvation at early morning.
These results indicate that long-term oral supplementation with a BCAA mixture is better than ordinary food in a late evening snack at improving the serum albumin level and the energy metabolism in patients with cirrhosis.
Fig. 2 Effect of BCAA mixture on serum albumin levels and fuel converstion
b. Nutrition assessment of bed-ridden patients
We studied the nutrition status of elderly bed-ridden patients. Muscle mass and serum albumin levels were significantly decreased compared to normal elderly people. Provision of higher energy and protein did not improve muscle mass or serum albumin levels. We postulate that immobilization decreases synthesis and increases breakdown of albumin and muscle protein. We are developing a nutrients which attenuate breakdown of muscle and albumin during immobilization.

We also studied effect of hypermetabolic state on nutritional status. During infection, muscle and albumin breakdown becomes greater because of cytokine and catecholamine. Cytokines decrease activity of lipoprotein lipase. We administered lipoprotein lipase activator, NO-1886, to the infection model animal. It improved appetite and weight loss, suggesting that this treatment is effective in malnourished patients.
3. Establishment of SPORTS (Spontaneously Running Tokushima-Shikoku) rat
The model animal for high level for voluntary wheel running was established in our department. Neurological mechanisms for wheel running of rodents, especially with high exercise motives would be applicable to the strategy for promotion of exercise motives in humans. Here we examined the regulation of norepinephrine (NE) system in the hippocampus of SPORTS rats.
In the hippocampus of SPORTS rats, the levels of NE in the extracellular fluid was augmented, whereas those of NE concentration in the whole homogenate of the tissue were decreased even in the sedentary conditions without running wheels. There was little change in the level of dopamine in the striatum. The protein expression and the activity levels of monoamine oxidase A (MAOA), a critical enzyme for the degradation of monoamines, were decreased in the hippocampus of SPORTS rats. In addition, the inhibition of MAOA activity in normal Wistar rats markedly increased wheel running activity. The hyper-running of SPORTS rats was suppressed by the treatment with clonidine, an agonist of a2 adrenergic receptor (AR), of which downregulation was also observed in the hippocampus of SPORTS rats.
Fig. 3 Wheel running distance of SPORTS rats
These results suggest that the elevation of extracellular NE and the possibly upstream or downstream depression of MAOA and a2 AR, respectively, in hippocampus determine the neural basis of the psychological regulation of exercise motives in the SPORTS rats. (Neuropsychopharmacology 2006; Life Sci 2005)
In addition to the running wheel, SPORTS rats showed high locomotion activity in the water immersion restraint stress, i.e. higher resistance to stress. Thus this rat is a good model to study stress coping effect (Fig.4).
Fig. 4 Immobilization during water immersion restraint stress in SPORTS rats and control rats
Fig. 5 A big thrombus in the left atrium which occupies the whole atirumThis rat also shows mild hypertension and produces thrombus in the left atrium, suggesting that this rat is a good model of formation of thrombus (Fig. 5)
References
  1. Chikahisa S, Sei H, Morishima M, Sano A, Kitaoka K, Nakaya Y, and Morita Y. Exposure to music in the perinatal period enhances learning performance and alters BDNF/TrkB signaling in mice as adults. Behav Brain Res 169: 312-319, 2006.
  2. Furukawa T, Bai CX, Kaihara A, Ozaki E, Kawano T, Nakaya Y, Awais M, Sato M, Umezawa Y, and Kurokawa J. Ginsenoside Re, a main phytosterol of Panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones. Mol Pharmacol 70: 1916-1924, 2006.
  3. Harada N, Hara S, Yoshida M, Zenitani T, Mawatari K, Nakano M, Takahashi A, Hosaka T, Yoshimoto K, and Nakaya Y. Molecular cloning of a murine glycerol-3-phosphate acyltransferase-like protein 1 (xGPAT1). Mol Cell Biochem 297: 41-51, 2007.
  4. Harada N, Kusuyama A, Morishima M, Okada K, Takahashi A, and Nakaya Y. Bezafibrate improves bacterial lipopolysaccharide-induced dyslipidemia and anorexia in rats. Metabolism 56: 517-522, 2007.
  5. Harada N, Ninomiya C, Osako Y, Morishima M, Mawatari K, Takahashi A, and Nakaya Y. Taurine alters respiratory gas exchange and nutrient metabolism in type 2 diabetic rats. Obes Res 12: 1077-1084, 2004.
  6. Hayabuchi Y, Nakaya Y, Yasui S, Mawatari K, Mori K, Suzuki M, and Kagami S. Angiotensin II activates intermediate-conductance Ca2+ -activated K+ channels in arterial smooth muscle cells. J Mol Cell Cardiol 41: 972-979, 2006.
  7. Hirasaka K, Kohno S, Goto J, Furochi H, Mawatari K, Harada N, Hosaka T, Nakaya Y, Ishidoh K, Obata T, Ebina Y, Gu H, Takeda S, Kishi K, and Nikawa T. Deficiency of Cbl-b gene enhances infiltration and activation of macrophages in adipose tissue and causes peripheral insulin resistance in mice. Diabetes, 2007.
  8. Kishi K, Muromoto N, Nakaya Y, Miyata I, Hagi A, Hayashi H, and Ebina Y. Bradykinin directly triggers GLUT4 translocation via an insulin-independent pathway. Diabetes 47: 550-558, 1998.
  9. Kusunoki M, Hara T, Tsutsumi K, Nakamura T, Miyata T, Sakakibara F, Sakamoto S, Ogawa H, Nakaya Y, and Storlien LH. The lipoprotein lipase activator, NO-1886, suppresses fat accumulation and insulin resistance in rats fed a high-fat diet. Diabetologia 43: 875-880, 2000.
  10. Li Z, Nakaya Y, Niwa Y, Chen X. K(Ca) channel-opening activity of Ginkgo Biloba extracts and ginsenosides in cultured endothelial cells. Clin Exp Pharmacol Physiol 28:441-5, 2001
  11. Manabe S, Kurroda I, Okada K, Morishima M, Okamoto M, Harada N, Takahashi A, Sakai K, and Nakaya Y. Decreased blood levels of lactic acid and urinary excretion of 3-methylhistidine after exercise by chronic taurine treatment in rats. J Nutr Sci Vitaminol 49: 375-380, 2003.
  12. Masori M, Hamamoto A, Mawatari K, Harada N, Takahasi A, and Nakaya Y. Angiotensin II decreases glucose uptake by downregulation of GLUT1 in the cell membrane of the vascular smooth muscle cell line A10. J Cardiovasc Pharmacol 50: 267-273, 2007.
  13. Matsushima R, Harada N, Webster NJ, Tsutsumi YM, and Nakaya Y. Effect of TRB3 on Insulin and Nutrient-stimulated Hepatic p70 S6 Kinase Activity. J Biol Chem 281: 29719-29729, 2006.
  14. Mawatari K, Kakui S, Harada N, Ohnishi T, Niwa Y, Okada K, Takahashi A, Izumi K, and Nakaya Y. Endothelin-1(1-31) levels hypercholesterolemic hamsters. Atherosclerosis 175: 203-212, 2004.
  15. Minami A, Iseki M, Kishi K, Wang M, Ogura M, Furukawa N, Hayashi S, Yamada M, Obata T, Takeshita Y, Nakaya Y, Bando Y, Izumi K, Moodie SA, Kajiura F, Matsumoto M, Takatsu K, Takaki S, and Ebina Y. Increased insulin sensitivity and hypoinsulinemia in APS knockout mice. Diabetes 52: 2657-2665, 2003.
  16. Morishima M, Harada N, Hara S, Sano A, Seno H, Takahashi A, Morita Y, and Nakaya Y. Monoamine oxidase A activity and norepinephrine level in hippocampus determine hyperwheel running in SPORTS rats. Neuropsychopharmacology 31: 2627-2638, 2006.
  17. Morishima-Yamato M, Hisaoka F, Shinomiya S, Harada N, Matoba H, Takahashi A, and Nakaya Y. Cloning and establishment of a line of rats for high levels of voluntary wheel running. Life Sci 77: 551-561, 2005.
  18. Nakano M, Takahashi A, Sakai Y, and Nakaya Y. Modulation of Pathogenicity with Norepinephrine Related to the Type III Secretion System of Vibrio parahaemolyticus. J Infect Dis 195: 1353-1360, 2007.
  19. Nakaya Y, Mawatari K, Takahashi A, Harada N, Hata A, and Yasui S. The phytoestrogen ginsensoside Re activates potassium channels of vascular smooth muscle cells through PI3K/Akt and nitric oxide pathways. J Med Invest 54: 381-384, 2007.
  20. Nakaya Y, Minami A, Harada N, Sakamoto S, Niwa Y, and Ohnaka M. Taurine improves insulin sensitivity in the Otsuka Long-Evans Tokushima Fatty rat, a model of spontaneous type 2 diabetes. Am J Clin Nutr 71: 54-58, 2000.
  21. Nakaya Y, Okita K, Suzuki K, Moriwaki H, Kato A, Miwa Y, Shiraishi K, Okuda H, Onji M, Kanazawa H, Tsubouchi H, Kato S, Kaito M, Watanabe A, Habu D, Ito S, Ishikawa T, Kawamura N, and Arakawa Y. BCAA-enriched snack improves nutritional state of cirrhosis. Nutrition 23: 113-120, 2007.
  22. Takahashi A, Miyoshi S, Takata N, Nakano M, Hamamoto A, Mawatari K, Harada N, Shinoda S, and Nakaya Y. Haemolysin produced by Vibrio mimicus activates two Cl2 secretory pathways in cultured intestinal-like Caco-2 cells. Cell Microbiol 9: 583-595, 2007.
  23. Takahashi A, Nakano M, Okamoto K, Fujii Y, Mawatari K, Harada N, and Nakaya Y. Aeromonas sobria hemolysin causes diarrhea by increasing secretion of HCO3. FEMS Microbiol Lett 258: 92-95, 2006.
  24. Takahashi A, Tanoue N, Nakano M, Hamamoto A, Okamoto K, Fujii Y, Harada N, and Nakaya Y. A pore-forming toxin produced by Aeromonas sobria activates Ca2+ dependent Cl- secretion. Microb Pathog 38: 173-180, 2005.

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