May 2017, The International Conference of Korean Society of Food Science of Animal Resources and 49th Annual Meeting, Postar award　　　　　　　　　　　　　　　
Ingestion of skim milk cheese modulates bile acid metabolism and cholic acid-induced disorders in rats
ISHIZUKA Satoshi

Nov. 2015, 6th International Conference on Food Factors, Postar award　　　　　　　　　　　　　　　
Isomaltomegalosaccharides modulation rat macrophage functions in primary culture and cytokine expression in rat intestinal mucosa
ISHIZUKA Satoshi

Nov. 2011, International Society for Nutraceuticals and Functional Foods, Poster award　　　　　　　　　　　　　　　
Dietary influence of linear α-1,6-polysaccharides on several parameters under experimental colitis depends on degree of the polymerization

May 2003, 日本栄養・食糧学会, 奨励賞　　　　　　　　　　　　　　　
消化管上皮の恒常性維持に関する基礎的研究
石塚 敏, Japan

Dietary supplementation with cholic acid reduces insulin secretion in response to intraperitoneal glucose administration in rats
Fumika Yokoyama, Reika Yoshitsugu, Hiromichi Yamazaki, Satoshi Ishizuka
Journal of Nutritional Science and Vitaminology, 70, 1, 76, 81, Feb. 2024, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal

Differences in iron balance observed with dietary cholic acid supplementation and marginal iron deficiency in rats.
Natsuki Kubota, Shota Hori, Satoshi Ishizuka
Bioscience, biotechnology, and biochemistry, 88, 1, 79, 85, 19 Dec. 2023, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, We investigated whether a cholic acid (CA)-supplemented diet and marginal iron deficiency (MID) diet influence hepatic lipid accumulation and iron balance in rats for 2 weeks. The CA diet enhanced hepatic lipid accumulation and modulated iron metabolism such as enhancement of fecal iron excretion, reduction in iron absorption, and no alteration in plasma iron levels. The MID diet did not alter hepatic lipid concentrations with reduced iron concentration in the liver and plasma. In combination, influence of the CA supplementation on the hepatic iron concentration was opposite between iron-sufficient and MID conditions. In the liver, the CA diet enhanced lipocalin 2 expression, whereas the MID diet enhanced transferrin receptor 1 expression and reduced hepcidin expression. This study revealed an involvement of 12-hydroxylated bile acids in regulation of hepatic iron concentration under MID condition.

An increase in enterohepatic 12-hydroxylated bile acid circulation and presymptomatic disorders　　　　　　　　　　　　　　　
Satoshi Ishizuka
Precision Medicine, 6, 14, 78, 81, Dec. 2023, [Invited], [Lead author, Corresponding author], [Domestic magazines]
Japanese, Scientific journal

White adipose tissue undergoes browning during preweaning period in association with microbiota formation in mice.
Anju Tsukada, Yuko Okamatsu-Ogura, Emi Futagawa, Yuki Habu, Natsumi Takahashi, Mira Kato-Suzuki, Yuko Kato, Satoshi Ishizuka, Kei Sonoyama, Kazuhiro Kimura
iScience, 26, 7, 107239, 107239, 21 Jul. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, Beige adipocytes are transiently induced during early postnatal period in mice. Previous studies have suggested that, unlike in adults, the induction is independent of the sympathetic nerve activity; however, the mechanism is yet unknown. Here, we showed that beige adipocytes are induced during the preweaning period in association with the formation of microbiota in mice. Alteration of gut microbiota composition in preweaning mice by maternal treatment with antibiotics or high-fat diet feeding substantially suppressed WAT browning. The suppression was also found in pups transplanted cecal microbiota from pups of high-fat diet-fed dams. These treatments reduced the hepatic expression of genes involved in bile acid synthesis and the serum bile acids level. The abundance of Porphyromonadaceae and Ruminococcaceae in microbiota showed a positive and negative correlation with the induction of beige adipocytes, respectively. This finding may provide comprehensive understanding of the association between gut microbiota and adipose tissue development in the neonatal period.

Supplementary effect of whey components on the monascin productivity of Monascus sp.
Qingyun Huang, Nodoka Miyaki, Zongfei Li, Yutaroh Takahashi, Satoshi Ishizuka, Toru Hayakawa, Jun-Ichi Wakamatsu, Haruto Kumura
Journal of the science of food and agriculture, 103, 8, 4234, 4241, Jun. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, BACKGROUND: Monascus sp. has been used in fermented foods for centuries. It can synthesize yellow, red, and orange pigments as secondary metabolites. Here, we focused on yellow pigment monascin, responsible for anti-inflammation and antidiabetic effects, and investigated whether whey could be a suitable substrate with or without rice powder for monascin production using M. purpureus AHU 9085, M. pilosus NBRC 4520 and M. ruber NBRC 32318. RESULTS: The growth and monascin production of the three Monascus strains were dependent on three liquid media consisting of whey and/or rice. All strains showed the best growth in a rice and whey mixed medium, in which M. ruber NBRC 32318 exhibited the highest total monascin production. Subsequent investigation of the effects of whey components indicated that a mineral cocktail in whey was particularly effective in stimulating the monascin production efficiency of M. ruber NBRC 32318. However, this recipe exhibited less stimulation, or even inhibition, for M. pilosus NBRC 4520 and M. purpureus AHU 9085, respectively. In terms of total monascin production, rice with whey provided the highest amount due to growth promotion along with relatively high production efficiency. CONCLUSION: The effect of whey on growth and monascin production was strongly dependent on the Monascus strains. Even a mineral cocktail in whey could regulate monascin productivity in a strain-specific manner. Further studies are needed to elucidate the mechanism behind the diverse responses by the minerals in the production of monascin from Monascus. © 2023 Society of Chemical Industry.

Ingesting a fermented milk product reduces liver triacylglycerol accumulation and normalizes gut permeability in rats even under a cholic acid-fed condition
DongGeun Lee, Wakana Iwasaki, Shota Hori, Natsuki Kubota, Satoshi Ishizuka
Journal of Functional Foods, 104, 105523, 105523, Elsevier BV, May 2023, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
Scientific journal

Metabolic alterations of the gut-liver axis induced by cholic acid contribute to hepatic steatosis in rats.
Hongxia Liu, Fumika Yokoyama, Satoshi Ishizuka
Biochimica et biophysica acta. Molecular and cell biology of lipids, 159319, 159319, 17 Apr. 2023, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, 12α-Hydroxylated (12αOH) bile acids (BAs) selectively increase with high-fat diet intake. Dietary supplementation with cholic acid (CA) in rats is a possible strategy to reveal the causal link between 12αOH BAs and hepatic steatosis. The present study aimed to investigate the metabolic mechanism underlying the effect of 12αOH BAs on hepatic steatosis. Male WKAH rats were fed either a control (Ct) or CA-supplemented diet (0.5 g/kg). After the 12-week intervention, the CA diet elevated the 12αOH BA levels in the gut-liver axis. CA-fed rats showed greater hepatic lipid accumulation than in the Ct group, regardless of the dietary energy balance. Untargeted metabolomics suggested marked differences in the fecal metabolome of rats subjected to the CA diet compared with that of Ct, characterized by the depletion of fatty acids and enrichment of amino acids and amines. Moreover, the liver metabolome differed in the CA diet group, characterized by an alteration in redox-related pathways. The CA diet elevated nicotinamide adenine dinucleotide consumption owing to the activation of poly(ADP-ribose) polymerase 1, resulting in impaired peroxisome proliferator-activated receptor α signaling in the liver. The CA diet increased sedoheptulose 7-phosphate, and enhanced glucose-6-phosphate dehydrogenase activity, suggesting promotion of the pentose phosphate pathway that generates reducing equivalents. Integrated analysis of the gut-liver metabolomic data revealed the role of deoxycholic acid and its liver counterpart in mediating these metabolic alterations. These observations suggest that alterations in metabolites induced by 12αOH BAs in the gut-liver axis contribute to the enhancement of liver lipid accumulation.

A diet supplemented with cholic acid elevates blood pressure accompanied by albuminuria in rats
Tomoko Shimoda, Hidehisa Shimizu, Wakana Iwasaki, Hongxia Liu, Yoshie Kamo, Koji Tada, Taketo Hanai, Shota Hori, Ga-Hyun Joe, Yasutake Tanaka, Masao Sato, Hitoshi Miyazaki, Satoshi Ishizuka
Bioscience, Biotechnology, and Biochemistry, 87, 4, 434, 441, Oxford University Press (OUP), Apr. 2023, [Peer-reviewed], [Last author, Corresponding author]
Scientific journal, Abstract

A diet supplemented with cholic acid (CA), the primary 12α-hydroxylated bile acid, can induce hepatic lipid accumulation in rats without obesity. This study examined the effects of a CA-supplemented diet on blood pressure (BP). After acclimation, WKAH/HkmSlc rats (3 week old) were divided into two groups and fed with a control AIN-93-based diet or a CA-supplemented diet (0.5 g CA/kg) for 13 weeks. The CA diet increased systolic and diastolic BP as well as hepatic lipid concentrations in the rats. No changes were found in the blood sodium concentration. Urinary albumin concentration increased in CA-fed rats. An increase was observed in the hepatic expression of ATP binding cassette subfamily B member 1B that correlated BPs and urinary albumin concentration accompanied by an increase in portal taurocholic acid concentration. These results suggest that 12α-hydroxylated bile acids are involved in increased BP and albuminuria via alteration of hepatic function.

Comparison of metabolism and circulation of 12α-hydroxylated bile acids in response to a high-fat diet　　　　　　　　　　　　　　　
Satoshi Ishizuka, Wakana Iwasaki
アグリバイオ, 6, 14, 60, 63, Dec. 2022, [Invited], [Lead author, Corresponding author], [Domestic magazines]
Japanese, Scientific journal

The ratio of 12α to non-12-hydroxylated bile acids reflects hepatic triacylglycerol accumulation in high-fat diet-fed C57BL/6J mice.
Wakana Iwasaki, Ryo Yoshida, Hongxia Liu, Shota Hori, Yuki Otsubo, Yasutake Tanaka, Masao Sato, Satoshi Ishizuka
Scientific reports, 12, 1, 16707, 16707, Oct. 2022, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, In our previous study, enterohepatic 12α-hydroxylated (12α) bile acid (BA) levels were found to be correlated with hepatic triacylglycerol concentration in rats fed high-fat (HF) diet. Since BA composition is diverse depending on animal species, we evaluated whether such a relationship is applicable in mice in response to an HF diet. C57BL/6JJmsSLC (B6) male mice were fed HF diet for 13 weeks and analyzed for triacylglycerol, cholesterol, oxysterols, and other metabolites in the liver. The BA composition was determined in the liver, small intestinal contents, portal plasma, aortic plasma, and feces. Neutral sterols were also measured in the feces. The ratio of 12α BA/non-12 BA increased in the liver, portal plasma, small intestinal contents, and feces of HF-fed B6 mice. Moreover, a positive correlation was observed between the ratio of fecal 12α BAs/non-12 BAs and hepatic triacylglycerol concentration. The concentration of 7α-hydroxycholesterol was increased in the liver of HF-fed B6 mice, whereas no increase was observed in the hepatic expression of cytochrome P450 family 7 subfamily A member 1. The present study showed that the ratio of 12α BA/non-12 BA in feces is closely associated with hepatic triacylglycerol accumulation in B6 mice fed HF diet.

生活習慣病の発症と12α水酸化胆汁酸：肝脂質蓄積と消化管バリア　　　　　　　　　　　　　　　
石塚 敏
Microbiome Science, 1, 1, 24, 29, Jul. 2022, [Invited], [Lead author, Corresponding author], [Domestic magazines]
Japanese, Scientific journal

Taurocholic acid, a primary 12α-hydroxylated bile acid, induces leakiness in the distal small intestine in rats.
Hongxia Liu, Ohji Kohmoto, Ayana Sakaguchi, Shota Hori, Misuzu Tochigi, Koji Tada, Yeonmi Lee, Keidai Kikuchi, Satoshi Ishizuka
Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 165, 113136, 113136, Jul. 2022, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, A high-fat diet increases 12α-hydroxylated (12αOH) bile acid (BA) secretion in rats, and secondary BAs are responsible for the leaky gut. This study aimed to examine the role of primary 12αOH BAs in gut barrier impairment in rats using dietary cholic acid (CA) supplementation (0.5 g/kg diet). The CA diet increased the 12αOH BAs concentrations in the small and large intestine, accompanied by gut barrier impairment. Based on the luminal 12αOH BAs concentrations, ex vivo gut leakiness was determined. Deoxycholic acid increased permeability in the large intestine, whereas taurocholic acid (TCA) increased the ileal permeability, but not jejunal permeability. A Rho kinase inhibitor attenuated TCA-induced ileal permeability. Administration of vancomycin, which abolishes secondary BAs, did not influence the gut leakiness induced by the CA diet. Changes in the gut permeation marker in the tail vein blood suggested the possibility that the CA-induced leakiness occurred in the small intestine. The CA diet enhanced the phosphorylation of myosin light chain 2 and reduced claudins expressions in rat ileal epithelia. Reductions in barrier function-related genes were observed in the ileum, but not in the colon of the CA-fed rats. Overall, the present study demonstrated the significance of TCA in proximal gut leakiness.

Dietary supplementation with okara and Bacillus coagulans lilac-01 improves hepatic lipid accumulation induced by cholic acids in rats
Yeonmi Lee, Yasutake Tanaka, Wakana Iwasaki, Fumika Yokoyama, Ga-Hyun Joe, Misaki Tsuji, Takuma Nose, Koji Tada, Taketo Hanai, Shota Hori, Hidehisa Shimizu, Kimiko Minamida, Kazunori Miwa, Satoshi Ishizuka
Journal of Functional Foods, 90, 104991, 104991, Elsevier BV, Mar. 2022, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, A dietary symbiotic supplement of okara, a by-product of tofu manufacturing with Bacillus coagulans lilac-01 found to reduce secondary bile acid (BA) in a rat study and cholic acid (CA), a primary 12α-hydroxylated (12αOH) BA, induces lipid accumulation in rats. In this study, we investigated whether dietary supplementation of a synbiotic with okara and Bacillus coagulans lilac-01 improves CA-induced hepatic lipid accumulation. Male Wistar/ST rats (4 weeks old) were fed with or without CA-supplementation combined with the synbiotic for 2 weeks. The CA diet increased hepatic triglycerides and the synbiotic diet in combination with CA normalized liver triglyceride concentration accompanied by suppression of hepatic fatty acid synthase expression, enhancement of fecal triglyceride excretion, and increase in fecal non-12αOH BA excretion. Dietary supplementation with the okara synbiotic ameliorated hepatic lipid accumulation probably by reducing de novo lipogenesis and enhancing fecal triglyceride excretion.

腸内での二次胆汁酸生成と腸内細菌叢変化・疾病の誘導　　　　　　　　　　　　　　　
吹谷 智, 横田 篤, 石塚 敏
機能性食品と薬理栄養, 15, 4, 229, 234, (株)インフォノーツパブリッシング, Feb. 2022, [Invited]
Japanese, Scientific journal

Dietary raffinose ameliorates hepatic lipid accumulation induced by cholic acid via modulation of enterohepatic bile acid circulation in rats.
Kenta Maegawa, Haruka Koyama, Satoru Fukiya, Atsushi Yokota, Koichiro Ueda, Satoshi Ishizuka
British journal of nutrition, 127, 11, 1621, 1630, 2022, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, Enterohepatic circulation of 12α-hydroxylated (12αOH) bile acid (BA) is enhanced depending on the energy intake in high-fat diet-fed rats. Such BA metabolism can be reproduced using a diet supplemented with cholic acid (CA), which also induces simple steatosis, without inflammation and fibrosis, accompanied by some other symptoms that are frequently observed in the condition of non-alcoholic fatty liver in rats. We investigated whether supplementation of the diet with raffinose (Raf) improves hepatic lipid accumulation induced by the CA-fed condition in rats. After acclimation to the AIN-93-based control diet, male Wistar rats were fed diets supplemented with a combination of Raf (30 g/kg diet) and/or CA (0·5 g/kg diet) for 4 weeks. Dietary Raf normalised hepatic TAG levels (two-way ANOVA P < 0·001 for CA, P = 0·02 for Raf and P = 0·004 for interaction) in the CA-supplemented diet-fed rats. Dietary Raf supplementation reduced hepatic 12αOH BA concentration (two-way ANOVA P < 0·001 for CA, P = 0·003 for Raf and P = 0·03 for interaction). The concentration of 12αOH BA was reduced in the aortic and portal plasma. Raf supplementation increased acetic acid concentration in the caecal contents (two-way ANOVA P = 0·001 as a main effect). Multiple regression analysis revealed that concentrations of aortic 12αOH BA and caecal acetic acid could serve as predictors of hepatic TAG concentration (R2 = 0·55, P < 0·001). However, Raf did not decrease the secondary 12αOH BA concentration in the caecal contents as well as the transaminase activity in the CA diet-fed rats. These results imply that dietary Raf normalises hepatic lipid accumulation via suppression of enterohepatic 12αOH BA circulation.

12α-Hydroxylated bile acid enhances accumulation of adiponectin and immunoglobulin A in the rat ileum.
Reika Yoshitsugu, Hongxia Liu, Yoshie Kamo, Akari Takeuchi, Ga-Hyun Joe, Koji Tada, Keidai Kikuchi, Nobuyuki Fujii, Shinri Kitta, Shota Hori, Manami Takatsuki, Hitoshi Iwaya, Yasutake Tanaka, Hidehisa Shimizu, Satoshi Ishizuka
Scientific reports, 11, 1, 12939, 12939, 21 Jun. 2021, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, We previously reported that dietary supplementation with cholic acid (CA), the primary 12α-hydroxylated (12αOH) bile acid (BA), reduces plasma adiponectin concentration in rats. The aim of this study was to examine the distribution of adiponectin in the body of CA-fed rats and its influence on mucosal immunoglobulin A concentration in the intestine. Rats were fed a diet supplemented with or without CA (0.5 g CA/kg diet) for 13 weeks. A reduction in plasma adiponectin level was observed from week 3. At the end of the experiment, the CA diet reduced plasma adiponectin concentration both in the portal and aortic plasma. Accumulation of adiponectin was accompanied by an increase in cadherin-13 mRNA expression in the ileal mucosa of CA-fed rats. No increase was observed in adiponectin mRNA expression in the ileal and adipose tissues of the CA-fed rats. Immunoglobulin A concentration in the ileal mucosa was elevated in the CA-fed rats and was correlated with the ileal adiponectin concentration. 12αOH BAs may modulate mucosal immune response that are involved in the accumulation of adiponectin in the ileum.

Primary 12α-Hydroxylated Bile Acids Lower Hepatic Iron Concentration in Rats.
Shota Hori, Minako Satake, Ohji Kohmoto, Ryo Takagi, Kazufumi Okada, Satoru Fukiya, Atsushi Yokota, Satoshi Ishizuka
The Journal of nutrition, 151, 3, 523, 530, 11 Mar. 2021, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, BACKGROUND: Primary 12α-hydroxylated bile acids (12αOH BAs) enhance intestinal iron uptake due to their ability ex vivo to chelate iron. However, no information is available on their role in vivo, especially in the liver. OBJECTIVES: To investigate the effects and mechanisms of primary 12αOH BAs on hepatic iron concentration in vivo. METHODS: Male Wistar King A Hokkaido male rats (WKAH/HkmSlc) rats aged 4-5 weeks were fed a control diet or a diet with cholic acid (CA; 0.5 g/kg diet), the primary 12αOH BA, for 2 weeks (Study 1) or 13 weeks (Study 2). In Study 3, rats fed the same diets were given drinking water either alone or containing vancomycin (200 mg/L) for 6 weeks. The variables measured included food intake (Studies 1-3), bile acid profiles (Studies 1 and 3), hepatic iron concentration (Studies 1-3), fecal iron excretion (Studies 1 and 2), iron-related liver gene expression (Studies 2 and 3), and plasma iron-related factors (Studies 2 and 3). RESULTS: In Study 1, CA feed reduced the hepatic iron concentration (-16%; P = 0.005) without changing food intake or fecal iron excretion. In Study 2, we found a significant increase in the aortic plasma concentration of lipocalin 2 (LCN2; +65%; P < 0.001), an iron-trafficking protein. In Study 3, we observed no effect of vancomycin treatment on the CA-induced reduction of hepatic iron concentration (-32%; P < 0.001), accompanied by increased plasma LCN2 concentration (+72%; P = 0.003), in the CA-fed rats despite a drastic reduction in the secondary 12αOH BA concentration (-94%; P < 0.001) in the aortic plasma. CONCLUSIONS: Primary 12αOH BAs reduced the hepatic iron concentration in rats. LCN2 may be responsible for the hepatic iron-lowering effect of primary 12αOH BAs by transporting iron out of the liver.

栄養過多による12α水酸化胆汁酸の増加とラットにおける肥満を伴わない肝脂質蓄積　　　　　　　　　　　　　　　
石塚 敏
生化学, 93, 1, 43, 51, Feb. 2021, [Peer-reviewed], [Invited], [Lead author, Corresponding author]
Japanese, Scientific journal

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats.
Ja-Young Lee, Hidehisa Shimizu, Masahito Hagio, Satoru Fukiya, Masamichi Watanabe, Yasutake Tanaka, Ga-Hyun Joe, Hitoshi Iwaya, Reika Yoshitsugu, Keidai Kikuchi, Misaki Tsuji, Nanako Baba, Takuma Nose, Koji Tada, Taketo Hanai, Shota Hori, Akari Takeuchi, Yumiko Furukawa, Bungo Shirouchi, Masao Sato, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota, Satoshi Ishizuka
Biochimica et biophysica acta. Molecular and cell biology of lipids, 1865, 12, 158811, 158811, 04 Sep. 2020, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.

A low coefficient of variation in hepatic triglyceride concentration in an inbred rat strain.
Tomoko Shimoda, Shota Hori, Kenta Maegawa, Akari Takeuchi, Yeonmi Lee, Ga-Hyun Joe, Yasutake Tanaka, Hidehisa Shimizu, Satoshi Ishizuka
Lipids in health and disease, 19, 1, 137, 137, 16 Jun. 2020, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, BACKGROUND: Inbred strains are characterized by less genetic variation, which suggests usefulness of inbred strains for evaluations of various parameters. In this study, experimental reproducibility in several parameters was compared between an outbred Wistar rat and Wistar King A Hokkaido (WKAH/HkmSlc) rat, the inbred strain that is originated from Wistar rats. METHODS: Difference of variations was investigated in parameters of body compositions and liver functions such as body weight, liver weight, liver triglycerides (TG), liver cholesterol and plasma alanine aminotransferase activity (ALT) between WKAH rats and outbred Wistar rats by using the coefficient of variation (CV). RESULTS: There was no difference in the CVs of body weight and relative liver weight between WKAH and Wistar rats. The CVs of body weight and relative liver weight were below 10% in both WKAH and Wistar rats. The CVs of TG, cholesterol, and ALT in Wistar rats were between 30 and 40%, whereas those in WKAH rats were between 10 and 25%. A low CV level of TG was observed in WKAH rats compared to that in Wistar rats regardless of the duration of the experimental period in those rat strains. CONCLUSION: The low CV values in metabolic parameters involved in liver functions in the inbred rats suggested an advantage of using inbred rather than outbred rats for the evaluation of liver lipid metabolism.

Lithocholic acid increases intestinal phosphate and calcium absorption in a vitamin D receptor dependent but transcellular pathway independent manner.
Nobuhiro Hashimoto, Isao Matsui, Satoshi Ishizuka, Kazunori Inoue, Ayumi Matsumoto, Karin Shimada, Shota Hori, Dong Geun Lee, Seiichi Yasuda, Yusuke Katsuma, Sachio Kajimoto, Yohei Doi, Satoshi Yamaguchi, Keiichi Kubota, Tatsufumi Oka, Yusuke Sakaguchi, Yoshitsugu Takabatake, Takayuki Hamano, Yoshitaka Isaka
Kidney international, 97, 6, 1164, 1180, Jun. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, Phosphate/calcium homeostasis is crucial for health maintenance. Lithocholic acid, a bile acid produced by intestinal bacteria, is an agonist of vitamin D receptor. However, its effects on phosphate/calcium homeostasis remain unclear. Here, we demonstrated that lithocholic acid increases intestinal phosphate/calcium absorption in an enterocyte vitamin D receptor-dependent manner. Lithocholic acid was found to increase serum phosphate/calcium levels and thus to exacerbate vascular calcification in animals with chronic kidney disease. Lithocholic acid did not affect levels of intestinal sodium-dependent phosphate transport protein 2b, Pi transporter-1, -2, or transient receptor potential vanilloid subfamily member 6. Everted gut sac analyses demonstrated that lithocholic acid increased phosphate/calcium absorption in a transcellular pathway-independent manner. Lithocholic acid suppressed intestinal mucosal claudin 3 and occludin in wild-type mice, but not in vitamin D receptor knockout mice. Everted gut sacs of claudin 3 knockout mice showed an increased permeability for phosphate, but not calcium. In patients with chronic kidney disease, serum 1,25(OH)2 vitamin D levels are decreased, probably as an intrinsic adjustment to reduce phosphate/calcium burden. In contrast, serum and fecal lithocholic acid levels and fecal levels of bile acid 7α-dehydratase, a rate-limiting enzyme involved in lithocholic acid production, were not downregulated. The effects of lithocholic acid were eliminated by bile acid adsorptive resin in mice. Thus, lithocholic acid and claudin 3 may represent novel therapeutic targets for reducing phosphate burden.

Association between 12α-hydroxylated bile acids and hepatic steatosis in rats fed a high-fat diet.
Shota Hori, Takayuki Abe, Dong Geun Lee, Satoru Fukiya, Atsushi Yokota, Nao Aso, Bungo Shirouchi, Masao Sato, Satoshi Ishizuka
The Journal of nutritional biochemistry, 83, 108412, 108412, 20 May 2020, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, High-fat (HF) diet induces hepatic steatosis that is a risk factor for noncommunicable diseases such as obesity, type 2 diabetes and cardiovascular disease. Previously, we found that HF feeding in rats increases the excretion of fecal bile acids (BAs), specifically 12α-hydroxylated (12αOH) BAs. Although the liver is the metabolic center in our body, the association between hepatic steatosis and 12αOH BAs in HF-fed rats is unclear. Thus, we investigated extensively BA composition in HF-fed rats and evaluated the association between hepatic steatosis and 12αOH BAs. Acclimated male inbred WKAH/HkmSlc rats were divided into two groups and fed either control or HF diet for 8 weeks. Feeding HF diet increased hepatic triglyceride and total cholesterol concentrations, which correlated positively with 12αOH BAs concentrations but not with non-12αOH BAs in the feces, portal plasma and liver. Accompanied by the increase in 12αOH BAs, the rats fed HF diet showed increased fat absorption and higher mRNA expression of liver Cidea. The enhancement of 12αOH BA secretion may contribute to hepatic steatosis by the promotion of dietary fat absorption and hepatic Cidea mRNA expression. The increase in 12αOH BAs was associated with enhanced liver cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) mRNA expression. There was a significant increase in 7α-hydroxycholesterol, a precursor of BAs, in the liver of HF-fed rats. Altogether, these data suggest that the HF diet increases preferentially 12αOH BAs synthesis by utilizing the accumulated hepatic cholesterol and enhancing mRNA expression of Cyp7a1 and Cyp8b1 in the liver.

Untargeted lipidomic analysis of plasma from high fat diet-induced obese rats using UHPLC- linear trap quadrupole -Orbitrap MS.
Siddabasave Gowda B Gowda, Zi-Jun Gao, Zhen Chen, Takayuki Abe, Shota Hori, Satoru Fukiya, Satoshi Ishizuka, Atsushi Yokota, Hitoshi Chiba, Shu-Ping Hui
Analytical sciences, 36, 7, 821, 828, 17 Jan. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, High-fat diet (HFD)-induced obesity is a primary risk factor for serious health problems. Although much research has been performed at the genomic level, lipidomic studies were limited. In this study, we aim to obtain a comprehensive profile of circulating plasma lipids, which are altered in rodent rat obesity by untargeted liquid chromatography-mass spectrometry. Rats fed with HFD for 8 weeks had increased body weight, liver and adipose tissue weight. The analysis results revealed that polyunsaturated fatty acids (PUFAs) and their corresponding phosphatidylcholine, phosphatidylinositol, and phosphatidylserine were significantly decreased in rats fed with HFD. In contrast, less unsaturated and ether type phosphatidylglycerols were increased. The triacylglycerides (TAGs) having saturated FA were increased in the HFD condition, whereas TAGs having PUFA were decreased. The levels of many plasma lipids were altered, and interestingly PUFA derived lipids were negatively associated with obesity. This signifies the importance of a PUFAs enriched diet to overwhelm obesity associated diseases.

Correlation between 12α-hydroxylated bile acids and insulin secretion during glucose tolerance tests in rats fed a high-fat and high-sucrose diet.
Reika Yoshitsugu, Keidai Kikuchi, Shota Hori, Hitoshi Iwaya, Masahito Hagio, Hidehisa Shimizu, Tohru Hira, Satoshi Ishizuka
Lipids in health and disease, 19, 1, 9, 9, 15 Jan. 2020, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, BACKGROUND: Previously, we found a significant relationship in a rat study between energy intake and bile acid (BA) metabolism especially 12α-hydroxylated (12αOH) BAs. The present study was designed to reveal relationships among BA metabolism, glucose tolerance, and cecal organic acids in rats fed a high-fat and high-sucrose diet (HFS) by using multivariate and multiple regression analyses in two types of glucose tolerance tests (GTTs). METHODS: Male WKAH/HkmSlc rats were fed with a control or a HFS for 13 weeks. Oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT) were performed at week 9 and 11, respectively. BAs were analyzed by using ultra high-performance liquid chromatography-mass spectrometry. Organic acid concentrations in cecal contents were analyzed by using ultra high-performance liquid chromatography with post-column pH buffered electric conductivity method. RESULTS: A positive correlation of aortic 12αOH BA concentration was observed with energy intake and visceral adipose tissue weight. We found that an increase of 12αOH BAs in enterohepatic circulation, intestinal contents and feces in the HFS-fed rats compared to those in control rats regardless of no significant increase of total BA concentration in the feces in the test period. Fecal 12αOH BA concentration was positively correlated with maximal insulin level in OGTT and area under curve of insulin in IPGTT. There was a positive correlation between aortic 12αOH BAs concentration and changes in plasma glucose level in both OGTT and IPGTT. In contrast, a decrease in the concentration of organic acids was observed in the cecal contents of the HFS-fed rats. Multiple linear regression analysis in the IPGTT revealed that the concentrations of aortic 12αOH BA and cecal acetic acid were the predictors of insulin secretion. Moreover, there was a positive correlation between concentration of portal 12αOH BAs and change in insulin concentration of peripheral blood in the IPGTT. CONCLUSION: The distribution analysis of BA compositions accompanied by GTTs revealed a close relationship between 12αOH BA metabolism and insulin secretion in GTTs in rats.

Ingestion of difructose anhydride III partially suppresses the deconjugation and 7α-dehydroxylation of bile acids in rats fed with a cholic acid-supplemented diet.
Dong Geun Lee, Shota Hori, Ohji Kohmoto, Shinri Kitta, Ryo Yoshida, Yasutake Tanaka, Hidehisa Shimizu, Keisuke Takahashi, Taizo Nagura, Hirokatsu Uchino, Satoru Fukiya, Atsushi Yokota, Satoshi Ishizuka
Bioscience, biotechnology, and biochemistry, 83, 7, 1329, 1335, Jul. 2019, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, Difructose anhydride III (DFAIII) is a prebiotic involved in the reduction of secondary bile acids (BAs). We investigated whether DFAIII modulates BA metabolism, including enterohepatic circulation, in the rats fed with a diet supplemented with cholic acid (CA), one of the 12α-hydroxylated BAs. After acclimation, the rats were fed with a control diet or a diet supplemented with DFAIII. After 2 weeks, each group was further divided into two groups and was fed diet with or without CA supplementation at 0.5 g/kg diet. BA levels were analyzed in aortic and portal plasma, liver, intestinal content, and feces. As a result, DFAIII ingestion reduced the fecal deoxycholic acid level via the partial suppression of deconjugation and 7α-dehydroxylation of BAs following CA supplementation. These results suggest that DFAIII suppresses production of deoxycholic acid in conditions of high concentrations of 12α-hydroxylated BAs in enterohepatic circulation, such as obesity or excess energy intake. Abbreviation: BA: bile acid; BSH: bile salt hydrolase; CA: cholic acid; DCA: deoxycholic acid; DFAIII: difructose anhydride III; MCA: muricholic acid; MS: mass spectrometry; NCDs: non-communicable diseases; LC: liquid chromatography; SCFA: short-chain fatty acid; TCA: taurocholic acid; TCDCA: taurochenodeoxycholic acid; TDCA: taurodeoxycholic acid; TUDCA: tauroursodeoxychlic acid; TαMCA: tauro-α-muricholic acid; TβMCA: tauro-β-muricholic acid; TωMCA: tauro-ω-muricholic acid.

Alteration of Bile Acid Metabolism by a High-Fat Diet Is Associated with Plasma Transaminase Activities and Glucose Intolerance in Rats.
Yoshitsugu R, Kikuchi K, Iwaya H, Fujii N, Hori S, Lee DG, Ishizuka S
Journal of Nutritional Science and Vitaminology, 65, 1, 45, 51, 2019, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]

Marginal iron deficiency enhances liver triglyceride accumulation in rats fed a high-sucrose diet.
Hori S, Hara H, Ishizuka S
Bioscience, Biotechnology, and Biochemistry, 82, 12, 2140, 2148, Dec. 2018, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]

Application of red pigment producing edible fungi for development of a novel type of functional cheese
Kumura Haruto, Ohtsuyama Takeru, Matsusaki Yoh-hey, Taitoh Miho, Koyanagi Haruka, Kobayashi Ken, Hayakawa Toru, Wakamatsu Jun-ichi, Ishizuka Satoshi
JOURNAL OF FOOD PROCESSING AND PRESERVATION, 42, 10, Oct. 2018, [Peer-reviewed]
English, Scientific journal

Influence of AHRR Pro189Ala polymorphism on kidney functions
Kazuhiro Nakayama, Shinichi Saito, Kazuhisa Watanabe, Hiroshi Miyashita, Fuyuhiko Nishijima, Yoshie Kamo, Koji Tada, Satoshi Ishizuka, Toshimitsu Niwa, Sadahiko Iwamoto, Hidehisa Shimizu
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 81, 6, 1120, 1124, TAYLOR & FRANCIS LTD, 2017, [Peer-reviewed]
English, Scientific journal, The function of aryl hydrocarbon receptor repressor (AHRR) in the kidney is unclear. The present study investigated associations between AHRR Pro189Ala polymorphism and estimated glomerular filtration rates (eGFR), serum creatinine, and hemoglobin levels in 2775 Japanese adults without diabetes. In addition, we examined whether AHRR expression levels in the kidney of control and chronic kidney disease (CKD) rats were changed. Multiple linear regression analyses showed that carriers of the Ala allele had increased eGFR and lower concentrations of serum creatinine and hemoglobin (p < 0.05). Immunohistochemical analysis showed that the expression of AHRR was upregulated in the kidneys of rats with CKD. These findings suggest that AHRR plays distinct roles in kidney functions and hemoglobin values. The effects of the AHRR polymorphism might be intensified in the kidneys of patients with CKD.

Combination of soya pulp and Bacillus coagulans lilac-01 improves intestinal bile acid metabolism without impairing the effects of prebiotics in rats fed a cholic acid-supplemented diet
Yeonmi Lee, Reika Yoshitsugu, Keidai Kikuchi, Ga-Hyun Joe, Misaki Tsuji, Takuma Nose, Hidehisa Shimizu, Hiroshi Hara, Kimiko Minamida, Kazunori Miwa, Satoshi Ishizuka
BRITISH JOURNAL OF NUTRITION, 116, 4, 603, 610, CAMBRIDGE UNIV PRESS, Aug. 2016, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Intestinal bacteria are involved in bile acid (BA) deconjugation and/ or dehydroxylation and are responsible for the production of secondary BA. However, an increase in the production of secondary BA modulates the intestinal microbiota due to the bactericidal effects and promotes cancer risk in the liver and colon. The ingestion of Bacillus coagulans improves constipation via the activation of bowel movement to promote defaecation in humans, which may alter BA metabolism in the intestinal contents. BA secretion is promoted with high-fat diet consumption, and the ratio of cholic acid (CA): chenodeoxycholic acid in primary BA increases with ageing. The dietary supplementation of CA mimics the BA environment in diet-induced obesity and ageing. We investigated whether B. coagulans lilac-01 and soya pulp influence both BA metabolism and the maintenance of host health in CA-supplemented diet-fed rats. In CA-fed rats, soya pulp significantly increased the production of secondary BA such as deoxycholic acid and omega-muricholic acids, and soya pulp ingestion alleviated problems related to plasma adiponectin and gut permeability in rats fed the CA diet. The combination of B. coagulans and soya pulp successfully suppressed the increased production of secondary BA in CA-fed rats compared with soya pulp itself, without impairing the beneficial effects of soya pulp ingestion. In conclusion, it is possible that a combination of prebiotics and probiotics can be used to avoid an unnecessary increase in the production of secondary BA in the large intestine without impairing the beneficial functions of prebiotics.

Physiological concentrations of trans-11 18:1 vaccenic acid suppress pro-inflammatory markers under acute inflammation in isolated ICR mice splenocytes
Jae-Sung Lee, Ji-Na Lim, Tao Wang, Sang-Bum Lee, Jin-Hee Hwang, U-Suk Jung, Min-Jeong Kim, Seong-Ho Choi, Satoshi Ishizuka, Hong-Gu Lee
FOOD SCIENCE AND BIOTECHNOLOGY, 25, 1, 275, 281, KOREAN SOCIETY FOOD SCIENCE & TECHNOLOGY-KOSFOST, Feb. 2016, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, Direct influences of dietary trans-11 18:1 vaccenic acid (TVA) at physiological concentrations of 50-200 mu M were evaluated for cell growth, cytotoxic activity, and cytokine production in leukocytes isolated from the mouse spleen. TVA supplementation for 24 h induced growth of splenocytes at concentrations of 50-200 mu M, except for 100 mu M. The cytokines TNF alpha, IFN gamma, and IL-10 of splenocytes were stimulated by 100 mu M TVA. Induced production of TNF alpha in splenocytes challenged with lipopolisaccharides was suppressed by 100 mu M TVA. Physiological levels of TVA had direct effects on growth and cytokine production in splenocytes. Further in vivo studies are needed to improve understanding of the precise influence of trans fatty acids on production of pro-inflammatory markers under acute inflammation conditions.

Megalo-type alpha-1,6-glucosaccharides induce production of tumor necrosis factor alpha in primary macrophages via toll-like receptor 4 signaling
Ga-Hyun Joe, Midori Andoh, Aki Shinoki, Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Masayuki Okuyama, Atsuo Kimura, Hidehisa Shimizu, Hiroshi Hara, Satoshi Ishizuka
BIOMEDICAL RESEARCH-TOKYO, 37, 3, 179, 186, BIOMEDICAL RESEARCH PRESS LTD, 2016, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, The term "megalo-saccharide" is used for saccharides with ten or more saccharide units, whereas the term "oligo-saccharide" is used for saccharides containing fewer than ten monosaccharide units. Megalo-type alpha-1,6-glucosaccharide (M-IM) is a non-digestible saccharide and not utilized by intestinal bacteria, suggesting that ingested M-IM may encounter ileum Peyer's patches that contains immune cells such as macrophages. Macrophages are responsible for antigen incorporation and presentation during the initial step of immune responses. We investigated whether M-IMs modulate macrophage functions such as cytokine production, nitric oxide production, cell viability, and phagocytosis. Primary macrophages collected from the rats were cultured with the existence of M-IM or lipopolysaccharides (LPS). M-IM and LPS induced the production of tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL6), and nitric oxide in the primary macrophages. The gene expression profile of inflammatory factors including TNF alpha, IL6, and IL1 beta in M-IM-stimulated cells was similar to that of LPS-stimulated cells. The M-IM did not affect phagocytosis in the primary macrophages. The M-IM-induced TNF alpha production was suppressed in the cells treated with a toll-like receptor 4 (TLR4) inhibitor called TAK-242. In conclusion, the M-IM modulates cytokine expression via TLR4 signaling and may play a role in the modulation of immune responses.

Activity of ERK regulates mucin 3 expression and is involved in undifferentiated Caco-2 cell death induced by 3-oxo-C-12-homoserine lactone
Hidehisa Shimizu, Nanako Baba, Takuma Nose, Ryoko Taguchi, Shinya Tanaka, Ga-Hyun Joe, Hideaki Maseda, Nobuhiko Nomura, Masahito Hagio, Ja-Young Lee, Satoru Fukiya, Atsushi Yokota, Satoshi Ishizuka, Hitoshi Miyazaki
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 79, 6, 937, 942, TAYLOR & FRANCIS LTD, Jun. 2015, [Peer-reviewed]
English, Scientific journal, The signal molecule, 3-oxo-C-12-homoserine lactone (3-oxo-C-12-HSL), is similar to a mammalian hormone in bacteria. Although most studies have examined the effects of high 3-oxo-C-12-HSL concentrations (>200 mu M) on mammalian cellular functions because ~600 mu M 3-oxo-C-12-HSL can be secreted in biofilms of Pseudomonas aeruginosa grown in vitro, we previously showed that a low 3-oxo-C-12-HSL concentration (30 mu M) induces the apoptosis of undifferentiated Caco-2 cells through suppressing Akt activity. Here, we found that a low concentration of 3-oxo-C-12-HSL-activated ERK1/2 in undifferentiated Caco-2 cells. Incubating cells with the ERK pathway inhibitor U0126 for 30min alleviated the mucin 3 (MUC3) expression suppressed by 3-oxo-C-12-HSL, and the upregulation of MUC3 expression induced by a 48-h incubation with U0126-reduced cell death. Thus, altered MUC3 expression caused by long-term attenuated ERK1/2 activity might correlate with the death of undifferentiated Caco-2 cells induced by 3-oxo-C-12-HSL.

Diet supplementation with cholic acid promotes intestinal epithelial proliferation in rats exposed to gamma-radiation
Masahito Hagio, Hidehisa Shimizu, Ga-Hyun Joe, Manami Takatsuki, Maiko Shiwaku, Hong Xu, Ja-Young Lee, Nobuyuki Fujii, Satoru Fukiya, Hiroshi Hara, Atsushi Yokota, Satoshi Ishizuka
TOXICOLOGY LETTERS, 232, 1, 246, 252, ELSEVIER IRELAND LTD, Jan. 2015, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Consumption of a high-fat diet increases some secondary bile acids (BAs) such as deoxycholic acid (DCA) in feces. DCA is derived from cholic acid (CA), a primary BA. We evaluated intestinal epithelial proliferation and BA metabolism in response to oral administration of cholic acid (CA) in rats to determine the influence of a CA diet on the responses of gut epithelia to gamma-rays. WKAH/HkmSlc rats were divided into two dietary groups: control diet or CA-supplemented (2 g/kg diet) diet. Some of the rats from each group were irradiated with gamma-rays, and epithelial cell proliferation in the colon was analyzed histochemically. Unirradiated CA-fed rats had high levels of DCA and CA in the sera, as well as the presence of taurocholic acid in their feces. Significant increases were observed in both epithelial proliferation and the number of epithelial cells in the colon of the CA-fed rats, and this effect was observed at 8 weeks after gamma-ray exposure. Furthermore, extracts from both cecal contents and sera of the unirradiated CA-fed rats promoted proliferation of IEC-6 cells. These results indicate that BAs in enterohepatic circulation promote proliferation and survival of the intestinal epithelium after receiving DNA damage. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

Deoxycholic Acid Is Involved in the Proliferation and Migration of Vascular Smooth Muscle Cells
Hidehisa Shimizu, Masahito Hagio, Hitoshi Iwaya, Ikuya Tsuneki, Ja-Young Lee, Satoru Fukiya, Atsushi Yokota, Hitoshi Miyazaki, Hiroshi Hara, Satoshi Ishizuka
JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY, 60, 6, 450, 454, CENTER ACADEMIC PUBL JAPAN, Dec. 2014, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Obesity is increasingly becoming associated with increased risk of atherosclerosis. Serum levels of the bile acid deoxycholic acid (DCA) are elevated in mice with obesity induced by a high-fat (HF) diet. Therefore, we investigated the influence of DCA on the functions of vascular smooth muscle cells (VSMCs) because the initiation and progression of atherosclerosis are associated with VSMC proliferation and migration. DCA induced c-jun N-terminal kinase (JNK) activation whereas a JNK inhibitor prevented DCA-induced VSMC proliferation and migration. Based on these findings, we examined whether DCA promotes the expression of platelet-derived growth factor beta-receptor (PDGFR beta) that has a c-Jun binding site in its promoter region. The mRNA and protein expression levels of PDGFR beta were upregulated in VSMCs after a 24- and 48-h incubation with DCA, respectively. The effects of PDGF such as proliferation and migration of VSMCs were promoted after a 48-h incubation with DCA despite the absence of DCA during PDGF stimulation. These findings suggest that elevated serum concentrations of DCA are involved in the pathogenesis of atherosclerosis in HF-induced obesity.

Mucin 3 is involved in intestinal epithelial cell apoptosis via N-(3-oxododecanoyl)-L-homoserine lactone-induced suppression of Akt phosphorylation
Ryoko Taguchi, Shinya Tanaka, Ga-Hyun Joe, Hideaki Maseda, Nobuhiko Nomura, Junji Ohnishi, Satoshi Ishizuka, Hidehisa Shimizu, Hitoshi Miyazaki
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 307, 2, C162, C168, AMER PHYSIOLOGICAL SOC, Jul. 2014, [Peer-reviewed]
English, Scientific journal, N-acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)- L-homoserine lactone (3-oxo-C-12-HSL). A low concentration of 3-oxo-C-12-HSL (30 mu M) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C-12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C-12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C-12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C-12-HSL. 3-Oxo-C-12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C-12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C-12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C-12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.

Acyl-homoserine lactones suppresses IEC-6 cell proliferation and increase permeability of isolated rat colon
Ga-Hyun Joe, Midori Andoh, Mikako Nomura, Hitoshi Iwaya, Jae-Sung Lee, Hidehisa Shimizu, Youhei Tsuji, Hideaki Maseda, Hitoshi Miyazaki, Hiroshi Hara, Satoshi Ishizuka
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 78, 3, 462, 465, TAYLOR & FRANCIS LTD, 2014, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, We investigated to determine whether a variety of acyl-homoserine lactones (AHLs) influences epithelial cell proliferation and mucosal permeability. 3-Oxo-C-12-homoserine lactone (HSL) and 3-oxo-C-14-HSL significantly suppressed IEC-6 cell proliferation. A significant increase in mucosal permeability was observed in isolated rat colon tissue exposed to C-12-HSL, 3-oxo-C-12-HSL, and 3-oxo-C-14-HSL. These data indicate that AHLs suppress epithelial proliferation and disrupt barrier function in intestinal mucosa.

Contribution of Dipeptidyl Peptidase IV to the Severity of Dextran Sulfate Sodium-Induced Colitis in the Early Phase
Hitoshi Iwaya, Nobuyuki Fujii, Masahito Hagio, Hiroshi Hara, Satoshi Ishizuka
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77, 7, 1461, 1466, TAYLOR & FRANCIS LTD, Jul. 2013, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Dipeptidyl peptidase IV (DPPIV) degrades some peptide hormones and cytokines, resulting in homeostatic modulation. However, the role of DPPIV in inflammatory bowel diseases remains controversial. To determine the role of DPPIV in colitis, we used F344/DuCrlCrlj (F344/Du) rats as a DPPIV-deficient model. The serum DPPIV activity was much lower in the F344/Du rats than in F344/Jcl rats which were used as a DPPIV-positive model. Interestingly, the disease activity index (DAI) was different in the early phase of 2% dextran sulfate sodium (DSS)-induced colitis, as judged by the mucosal myeloperoxidase activity, colonic weight, and cecal fermentation. Similarly, retarded DAI was apparent in the DPPIV-deficient rats with 1% DSS-induced colitis. These findings suggest that a low level of DPPIV activity contributed to maintaining intestinal homeostasis by suppressing the cleavage of cytokines and growth hormones in DSS-induced colitis, especially in the early phase of colitis and with moderate inflammation.

A novel mechanism for the promotion of quercetin glycoside absorption by megalo alpha-1,6-glucosaccharide in the rat small intestine
Aki Shinoki, Weeranuch Lang, Charin Thawornkuno, Hee-Kwon Kang, Yuya Kumagai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka, Hiroshi Hara
FOOD CHEMISTRY, 136, 2, 293, 296, ELSEVIER SCI LTD, Jan. 2013, [Peer-reviewed]
English, Scientific journal, The presence of an alpha-1,6-glucosaccharide enhances absorption of water-soluble quercetin glycosides, a mixture of quercetin-3-O-beta-D-glucoside (Q3G, 31.8%), mono (23.3%), di (20.3%) and more D-glucose adducts with alpha-1,4-linkage to a D-glucose moiety of Q3G, in a ligated small intestinal loop of anesthetized rats. We prepared alpha-1,6-glucosaccharides with different degrees of polymerization (DP) enzymatically and separated them into a megalo-isomaltosaccharide-containing fraction (M-IM, average DP = 11.0) and an oligo-isomaltosaccharide-containing fraction (O-IM, average DP = 3.6). Luminal injection of either saccharide fraction promoted the absorption of total quercetin-derivatives from the small intestinal segment and this effect was greater for M-IM than O-IM addition. M-IM also increased Q3G, but not the quercetin aglycone, concentration in the water-phase of the luminal contents more strongly than O-IM. The enhancement of Q3G solubilization in the luminal contents may be responsible for the increases in the quercetin glucoside absorption promoted by alpha-1,6-glucosaccharides, especially that by M-IM. These results suggest that the ingestion of alpha-1,6-glucosaccharides promotes Q3G bioavailability. (C) 2012 Elsevier Ltd. All rights reserved.

Zinc deficiency induces dysregulation of cytokine productions in an experimental colitis of rats
Erwin Suwendi, Hitoshi Iwaya, Jae-Sung Lee, Hiroshi Hara, Satoshi Ishizuka
BIOMEDICAL RESEARCH-TOKYO, 33, 6, 329, 336, BIOMEDICAL RESEARCH PRESS LTD, Dec. 2012, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Dextran sulfate sodium (DSS)-induced colitis is an experimental model of ulcerative colitis, although the precise mechanism has not yet been elucidated. We investigate whether Zn deficiency affects the pathogenesis of colitis induced by DSS with a focus on immune responses. Male WKAH/Hkm Sic rats were fed either a Zn-adequate (ZA, 30 mg Zn/kg diet) as a control or Zn-deficient (ZD, 5 mg Zn/kg diet) diet for 21 days and then treated with 2% DSS via deionized drinking water for 7 days. The disease activity index (DAI) was recorded daily throughout DSS treatment. Serum Zn concentrations were significantly lowered in rats fed the ZD diet than those fed the ZA diet at day 7 and 14. Surprisingly, DSS treatment considerably reduced the serum Zn in both groups. The rats fed the ZD diet showed exacerbated colitis based on clinical outcomes, including weight loss, increased DAI, and shortened colon length. An in vitro study corroborated these results, showing that a large amount of TNF alpha was induced by rat mesenteric leukocytes in response to lipopolysaccharide in ZD medium, but not in ZA medium. These results indicate that a modulation of TNF alpha production due to Zn deficiency influences disease activity in DSS-induced colitis. In addition, more attention should be given to Zn for prevention of colitis.

The Delay in the Development of Experimental Colitis from Isomaltosyloligosaccharides in Rats Is Dependent on the Degree of Polymerization
Hitoshi Iwaya, Jae-Sung Lee, Shinya Yamagishi, Aki Shinoki, Weeranuch Lang, Charin Thawornkuno, Hee-Kwon Kang, Yuya Kumagai, Shiho Suzuki, Shinichi Kitamura, Hiroshi Hara, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka
PLOS ONE, 7, 11, e50658, PUBLIC LIBRARY SCIENCE, Nov. 2012, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Background: Isomaltosyloligosaccharides (IMO) and dextran (Dex) are hardly digestible in the small intestine and thus influence the luminal environment and affect the maintenance of health. There is wide variation in the degree of polymerization (DP) in Dex and IMO (short-sized IMO, S-IMO; long-sized IMO, L-IMO), and the physiological influence of these compounds may be dependent on their DP.
Methodology/Principal Findings: Five-week-old male Wistar rats were given a semi-purified diet with or without 30 g/kg diet of the S-IMO (DP = 3.3), L-IMO (DP = 8.4), or Dex (DP = 1230) for two weeks. Dextran sulfate sodium (DSS) was administered to the rats for one week to induce experimental colitis. We evaluated the clinical symptoms during the DSS treatment period by scoring the body weight loss, stool consistency, and rectal bleeding. The development of colitis induced by DSS was delayed in the rats fed S-IMO and Dex diets. The DSS treatment promoted an accumulation of neutrophils in the colonic mucosa in the rats fed the control, S-IMO, and L-IMO diets, as assessed by a measurement of myeloperoxidase (MPO) activity. In contrast, no increase in MPO activity was observed in the Dex-diet-fed rats even with DSS treatment. Immune cell populations in peripheral blood were also modified by the DP of ingested saccharides. Dietary S-IMO increased the concentration of n-butyric acid in the cecal contents and the levels of glucagon-like peptide-2 in the colonic mucosa.
Conclusion/Significance: Our study provided evidence that the physiological effects of alpha-glucosaccharides on colitis depend on their DP, linkage type, and digestibility. Citation: Iwaya H, Lee J-S, Yamagishi S, Shinoki A, Lang W, et al. (2012) The Delay in the Development of Experimental Colitis from Isomaltosyloligosaccharides in Rats Is Dependent on the Degree of Polymerization. PLoS ONE 7(11): e50658. doi: 10.1371/journal.pone.0050658

Glycochenodeoxycholic acid promotes proliferation of intestinal epithelia via reduction of cyclic AMP and increase in H2AX phosphorylation after exposure to gamma-rays
Satoshi Ishizuka, Maiko Shiwaku, Masahito Hagio, Takuya Suzuki, Tohru Hira, Hiroshi Hara
BIOMEDICAL RESEARCH-TOKYO, 33, 3, 159, 165, BIOMEDICAL RESEARCH PRESS LTD, Jun. 2012, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, Bile acids (BAs) are considered to be promotive factors in colorectal carcinogenesis. We investigated whether BAs in the cellular environment influence proliferation of intestinal epithelial cell lines. Some BAs induced proliferation in several epithelial cell lines. In the proliferation assay, significant increases in IEC-6 cell proliferation were observed in response to glycodeoxycholic acid or glycochenodeoxycholic acid (GCDCA). Among the glycine-conjugated derivatives of BAs, especially GCDCA reduced cAMP production in IEC-6 cells. Pertussis toxin completely inhibited the GCDCA-induced increase in IEC-6 proliferation, suggesting GCDCA-induced proliferation required G alpha i activation and cAMP reduction. Treatment with 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, also suppressed GCDCA-induced IEC-6 proliferation. We confirmed an increase in MEK1/2 phosphorylation in GCDCA-treated IEC-6 cells, and inhibition of MEK1/2 by U0126 clearly suppressed GCDCA-induced IEC-6 cell proliferation. A significant increase was observed in the phosphorylation of histone H2AX in GCDCA-treated IEC-6 cells after exposure to gamma-rays. Cell cycle analysis revealed that GCDCA increased the proportion of cells in S phase only after gamma-ray exposure. These results indicate that glycine-conjugated BAs in the cellular environment are potent inducers of cell proliferation accompanied by genomic instability in intestinal epithelia.

Mucosal permeability is an intrinsic factor in susceptibility to dextran sulfate sodium-induced colitis in rats
Hitoshi Iwaya, Koushi Maeta, Hiroshi Hara, Satoshi Ishizuka
EXPERIMENTAL BIOLOGY AND MEDICINE, 237, 4, 451, 460, ROYAL SOC MEDICINE PRESS LTD, Apr. 2012, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, We investigated differences in the pathogenesis of dextran sulfate sodium (DSS)-induced colitis between two inbred rat strains, Wistar King A Hokkaido (WKAH) and Dark Agouti (DA) rats, to determine the intrinsic factors responsible for the development of colitis. DSS exposure exacerbated the clinical symptoms such as body weight loss, stool consistency and rectal bleeding in DA rats rather than that in WKAH rats. Additionally, the average survival was shorter in DA rats than in WKAH rats. The expression levels of tumor necrosis factor-alpha, interleukin (IL)-12 p35 and IL-23 p19 increased prominently in the DA rats that were administered DSS, accompanied by severe infiltration of leukocytes into the colon. We also found that colonic permeability was greater in the DA rats than in the WKAH rats. In Ussing chambers, exposure of the isolated colon tissue to DSS enhanced the colonic permeability of both strains. Immunoblot analysis revealed that the expression levels of tight junction (TJ) proteins were modulated during DSS administration. Higher expression levels of claudin-4 and junctional adhesion molecule-A proteins were observed in DA rats than in WKAH rats, even in intact conditions. These results indicated that the expression pattern of TJ proteins determines the colonic permeability of the rats. In conclusion, the intrinsic colonic permeability is one of critical factors responsible for the susceptibility of rats to colitis.

Is bile acid a determinant of the gut microbiota on a high-fat diet?
Atsushi Yokota, Satoru Fukiya, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Satoshi Ishizuka
Gut Microbes, 3, 5, 455, 459, Landes Bioscience, 2012, [Peer-reviewed], [Last author]
English, Scientific journal, Recently, we discovered that bile acid, a main component of bile, is a host factor that regulates the composition of the cecal microbiota in rats. Because bile secretion increases on a high-fat diet and bile acids generally have strong antimicrobial activity, we speculated that bile acids would be a determinant of the gut microbiota in response to a high-fat diet. The observed changes in the rat cecal microbiota triggered by cholic acid (the most abundant bile acid in human biliary bile) administration resemble those found in animals fed high-fat diets. Here, we discuss the rationale for this hypothesis by evaluating reported diet-induced gut microbiota alterations based on the postulate that bile acids worked as an underlying determinant. The identification of host factors determining the gut microbiota greatly contributes to understanding the causal relationships between changes in the gut microbiota and disease development, which remain to be elucidated. © 2012 Landes Bioscience.

Bile Acid Is a Host Factor That Regulates the Composition of the Cecal Microbiota in Rats
K. B. M. Saiful Islam, Satoru Fukiya, Masahito Hagio, Nobuyuki Fujii, Satoshi Ishizuka, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota
GASTROENTEROLOGY, 141, 5, 1773, 1781, W B SAUNDERS CO-ELSEVIER INC, Nov. 2011, [Peer-reviewed]
English, Scientific journal, BACKGROUND & AIMS: Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. METHODS: Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. RESULTS: Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7 alpha-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. CONCLUSIONS: Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota.

Marginal Zinc Deficiency Exacerbates Experimental Colitis Induced by Dextran Sulfate Sodium in Rats
Hiroshi Iwaya, Munenori Kashiwaya, Aki Shinoki, Jae-Sung Lee, Kumiko Hayashi, Hiroshi Hara, Satoshi Ishizuka
JOURNAL OF NUTRITION, 141, 6, 1077, 1082, AMER SOC NUTRITIONAL SCIENCE, Jun. 2011, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, We investigated the impact of Zn status on the maintenance of mucosal homeostasis. Rats were fed diets containing different amounts of Zn (30, 10, 5, <1 mg Zn/kg diet) for 21 d. Serum Zn concentrations were lower in rats fed marginally Zn-deficient (MZD; 5 mg Zn/kg diet) and severely Zn-deficient (<1 mg/kg) diets but not in those fed the marginally Zn-adequate diet (10 mg/kg) or the Zn-adequate (ZA; 30 mg/kg) group (P < 0.05). However, organ weights, colonic epithelial cell proliferation, and crypt fission did not differ between the MZD and ZA groups. We then evaluated whether MZD modulated dextran sulfate sodium (DSS)-induced colonic inflammation by administering 2% DSS to the MZD and ZA groups for 7 d. Myeloperoxidase activity and TNF alpha production increased in response to DSS in the MZD group (P < 0.03). Colonic permeability in the 2 groups did not differ after DSS administration. In a culture experiment using isolated mesenteric leukocytes. TNF alpha production was higher (P < 0.05) and TNF receptor type I (TNFR1) expression was detected in culture medium containing 20 and 30 mu mol/L of Zn compared with culture medium lacking Zn supplementation. These results suggest that MZD exacerbated colitis by modulating the immune response through the impairment of TNF alpha production and TNFR1 expression rather than through the impairment of epithelial barrier function. J. Nutr. 141: 1077-1082, 2011.

Immunomodulatory effect of mushrooms on cytotoxic activity and cytokine production of intestinal lamina propria leukocytes does not necessarily depend on beta-glucan contents
Jae-Sung Lee, Kohsuke Oka, Osamu Watanabe, Hiroshi Hara, Satoshi Ishizuka
FOOD CHEMISTRY, 126, 4, 1521, 1526, ELSEVIER SCI LTD, Jun. 2011, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, We evaluated the effects of seven mushroom extracts (Grifola frondosa, Pholiota nameko, Panellus serotinus, Hypsizygus marmoreus, Pleurotus cornucopiae, Armillaria mellea, and Flammulina velutipes) on cytotoxic activity and cytokine production of lamina propria leukocytes (LPLs) isolated from rat small (S) and large (L) intestinal mucosa. Boiling water extracts from seven species of mushrooms showed no direct cytotoxicity against the YAC-1 target cells. However, prominent increases of cytotoxicity were observed in S- and L-LPLs co-cultured with P. serotinus extract. Cytokine production (TNF alpha, IFN gamma, IL-12 p70, and IL-4) of S- and L-LPLs was stimulated in response to P. cornucopiae extract. Mushroom extracts contributed to target cell adhesion and/or cytokine production in the effector cells. The promotion of cytotoxic activity in S- and L-LPLs was not necessarily related to beta-glucan content of the mushroom. (C) 2010 Elsevier Ltd. All rights reserved.

A partially degraded product of phytate suppresses the proliferation of HCT116 colorectal cancer cells
Satoshi Ishizuka, Ken-ichi Saitoh, Takuya Suzuki, Jae-Sung Lee, Hiroshi Hara
FOOD CHEMISTRY, 125, 4, 1219, 1225, ELSEVIER SCI LTD, Apr. 2011, [Peer-reviewed], [Lead author]
English, Scientific journal, Phytate myo inositol hexaphosphate (IP6) has been shown to possess anti-carcinogenic qualities IP6 appears to be partially digested in the digestive tract and degraded into inositol di tri- and tetraphosphate (IP2 IP3 and IP4) The present study investigated whether IP3-rich phytate hydrolysate (IP3-RPH) a partially degraded product from phytate plays a role in the suppression of cell proliferation using an HCT116 colon carcinoma cell line IP3-RPH suppresses cell proliferation as does IP6 and the suppression activity of IP3-RPH is higher than that of IP6 Suppression modes clearly differed between IP3-RPH and IP6 Our findings suggest that partially degraded IP6 products are responsible for the suppression of colon carcinogenesis by IP6 (C) 2010 Elsevier Ltd All rights reserved

Bile acid analysis in various biological samples using ultra performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS).
Hagio M, Matsumoto M, Ishizuka S
Methods in Molecular Biology (Clifton, N.J.), 708, 119, 129, 2011, [Peer-reviewed], [Invited], [Last author, Corresponding author]

Inulin Prolongs Survival of Intragastrically Administered Lactobacillus plantarum No. 14 in the Gut of Mice Fed A High-Fat Diet
Naoki Takemura, Masahiro Hagio, Satoshi Ishizuka, Hiroyuki Ito, Tatsuya Morita, Kei Sonoyama
JOURNAL OF NUTRITION, 140, 11, 1963, 1969, AMER SOC NUTRITIONAL SCIENCE, Nov. 2010, [Peer-reviewed]
English, Scientific journal, We tested whether a high-fat diet (HFD) impairs the survival of probiotics in mice. In Expt. 1, after feeding either a HFD (62.7% energy) or a normal-fat diet (NFD; 11.1% energy) for 2 d, C57BL16 mice were i.g. administered Lactobacillus plantarum No. 14. Fecal recovery of viable L. plantarum was significantly decreased 99% by the HFD compared with the NFD. Total bile acid concentrations in the small intestine and cecum were significantly higher 11.5- and 2.2-fold of NFD, respectively) in mice fed HFD than in those fed NFD. Cholic acid and deoxycholic acid significantly reduced the viability of L. plantarum No. 14 in culture experiments. In Expt. 2, after feeding HFD for 2 d, simultaneous administration of inulin 110 mg) with L. plantarum No. 14 significantly increased (100-fold of that without inulin) the fecal recovery of viable L. plantarum. Inulin administration did not alter intestinal bile acid concentrations. In Expt. 3, after feeding HFD for 2 d, mice were i.g. administered either inulin (10 mg) or vehicle and, after 6 h, cecal contents were subjected to culture experiments. Growth of L. plantarum No. 14 was significantly higher in the cecal contents of inulin-administered mice than vehicle-administered mice. Inulin supplementation to cecal contents of vehicle-administered mice significantly enhanced the growth of L. plantarum No. 14. We propose that HFD impairs the survival of probiotics in the gut due to increased bile acid stress and that simultaneous administration of inulin prolongs the survival of probiotics in mice fed HFD. J. Nutr. 140: 1963-1969, 2010.

Voluntary wheel running exercise and dietary lactose concomitantly reduce proportion of secondary bile acids in rat feces
Masahito Hagio, Megumi Matsumoto, Takaji Yajima, Hiroshi Hara, Satoshi Ishizuka
JOURNAL OF APPLIED PHYSIOLOGY, 109, 3, 663, 668, AMER PHYSIOLOGICAL SOC, Sep. 2010, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Hagio M, Matsumoto M, Yajima T, Hara H, Ishizuka S. Voluntary wheel running exercise and dietary lactose concomitantly reduce proportion of secondary bile acids in rat feces. J Appl Physiol 109: 663-668, 2010. First published July 8, 2010; doi: 10.1152/japplphysiol.00777.2009.-According to epidemiologic studies, a negative correlation exists between exercise amount and subsequent cancer development risk in the large intestine. The proportion of secondary bile acids (SBA) in the large intestine is related to subsequent risk for colorectal carcinogenesis. The aim of this study was to investigate the effects of voluntary wheel running exercise and dietary intervention on bile acid (BA) metabolism in the large intestine. Wistar/ST rats (6 wk old) were divided into two groups, exercise and sedentary, after acclimation. Four days after the animals were assigned to a group, rats in each group were fed diets supplemented with different carbohydrate sources including dextrin, sucrose, and lactose. The wheel running period was 4 wk in the exercise group, whereas rats in the sedentary group remained in individual cages during this period. BA composition in collected feces was analyzed with ultraperformance liquid chromatography-electrospray ionization mass spectrometry. We found that wheel running exercise decreased plasma concentrations of cholesterol, triglyceride, and free fatty acids. These decreases were accompanied by a reduction in the proportion of SBA to primary BA (PBA) in feces; however, daily excretion of BA was comparable regardless of wheel running exercise. In addition, ingestion of lactose decreased the SBA-to-PBA ratio and suppressed production of hyodeoxycholic acid in feces. In conclusion, voluntary wheel running exercise, in combination with dietary intervention, could independently reduce the SBA-to-PBA ratio within the large intestine without changing BA excretion. These changes may contribute to the prevention of colorectal carcinogenesis.

A novel mechanism underlying phytate-mediated biological action-phytate hydrolysates induce intracellular calcium signaling by a G alpha q protein-coupled receptor and phospholipase C-dependent mechanism in colorectal cancer cells
Takuya Suzuki, Teruhiro Nishioka, Satoshi Ishizuka, Hiroshi Hara
MOLECULAR NUTRITION & FOOD RESEARCH, 54, 7, 947, 955, WILEY-BLACKWELL, Jul. 2010, [Peer-reviewed]
English, Scientific journal, Phytate (inositol hexa-phosphate, IP6) possesses multiple biological functions including anticancer activity. IP6 is converted to inositol di-, tri-, and tetra-phosphates (IP2, IP3, and IP4) by phytase in large intestinal microbes; however, their contribution to the IP6-mediated functions has not been investigated. We have developed the preparations of IP2-4 and IP3-rich phytate hydrolysate (IP3-RPH) by IP6 digestion using microbial phytase, and examined the induction of intracellular Ca(2+) signaling in response to the preparations in colorectal cancer cells. IP2-4, but not inositol (IP0) and IP6, induced increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in Caco-2 cells with the following rank order: IP3 > IP2 - IP4. Inositol tri-phosphate (IP3)-RPH induced increases in [Ca(2+)](i) in both undifferentiated Caco-2 and HT-29 cells, but not in differentiated Caco-2. The IP3-RPH-induced [Ca(2+)](i) increase was resistant to extracellular Ca(2+) depletion, however, it was impaired by inhibitors of phospholipase C, inositol 1, 4, 5 tri-phosphate receptor, ryanodine receptor, and G alpha q protein. These results show that the putative G protein-coupled receptor on the plasma membrane senses the IP6 hydrolysates and activates phospholipase C beta, resulting in Ca(2+) mobilization through Ca(2+) channels coupled with the inositol 1, 4, 5 tri-phosphate and ryanodine receptors on the sarco-endoplasmic reticulum Ca(2+) store in colorectal cancer cells.

Improved Isolation Methods for Mucosal Leukocytes from Small and Large Intestines in Rats
Jae-Sung Lee, Kohsuke Oka, Mie Obara, Megumi Nishimukai, Yung-Choon Yoo, Kaori Yamada, Takamitsu Tsukahara, Keizo Nakayama, Hiroshi Hara, Satoshi Ishizuka
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73, 8, 1732, 1740, TAYLOR & FRANCIS LTD, Aug. 2009, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］
English, Scientific journal, We optimized the isolation protocol for intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from the rat small intestine, and LPLs from even the rat large intestine. The major population of IELs in the small intestine was considered to be from the villus epithelia. The cytotoxicity of mucosal leukocytes was comparable among isolated fractions from both the small and large intestines, regardless of the population differences. Further analyses of the cells collected from other lymphoid tissues demonstrated that CD161(+) cells selectively accumulated in the intestinal lamina propria and did not recirculate through the lymph ducts. Our modified isolation protocol enables the collection of mucosal immune cells from the rat intestines without any deterioration of cell function and could contribute to a better understanding of dietary influences on the mucosal immune system.

Administration of anti-glucagon-like peptide-2 serum suppresses epithelial cell proliferation of the distal small intestine in weanling rats
Satoshi Ishizuka, Ayako Inafune, Tohru Hira, Hirohisa Izumi, Kazuhiro Ozawa, Mitsunori Takase, Hiroshi Hara
BIOMEDICAL RESEARCH-TOKYO, 30, 4, 259, 261, BIOMEDICAL RESEARCH PRESS LTD, Aug. 2009, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, We investigated the effects of endogenous glucagon-like peptide-2 (GLP-2) on the development of intestinal mucosa in weanling rats. Three-week-old male weanling Sprague-Dawley rats were administered either anti-GLP-2 or normal rabbit serum every other day for 2 weeks. We then measured length, weight, and bromodeoxyuridine incorporation in the intestine on day 13 following the first injection. Administration of anti-GLP-2 serum significantly inhibited both epithelial proliferation in the distal ileum and elongation of the small intestine. These results Suggest that intrinsic GLP-2 contributes to the growth of the small intestine during the weanling period.

alpha-Lactalbumin Hydrolysate Stimulates Glucagon-Like Peptide-2 Secretion and Small Intestinal Growth in Suckling Rats
Hirohisa Izumi, Satoshi Ishizuka, Ayako Inafune, Tohru Hira, Kazuhiro Ozawa, Takashi Shimizu, Mitsunori Takase, Hiroshi Hara
JOURNAL OF NUTRITION, 139, 7, 1322, 1327, AMER SOC NUTRITIONAL SCIENCE, Jul. 2009, [Peer-reviewed]
English, Scientific journal, We investigated whether bovine milk constituents influenced glucagon-like peptide (GLP)-2 secretion and intestinal growth in suckling rats. Male Sprague-Dawley rats (14 d old) received i.g. infusions of a milk protein fraction, a lactose solution, or the cream fraction of milk. The serum concentration of GLP-2, but not GLP-1, markedly increased in rats administered milk protein compared with those given the lactose solution or the cream fraction from 60 to 120 min after administration. In another experiment, both casein (CN) and whey protein isolate stimulated GLP-2 secretion at 120 min after administration, but soy protein and ovalbumin did not. Stimulation of GLP-2 secretion by several milk proteins was similar, including alpha-CN, alpha-lactalbumin (alpha-La), and beta-lactoglobulin, in a separate experiment. A hydrolysate of alpha-La obtained by incubation with protease A extracted from Aspergillus oryzae (LaHPA) caused almost twice the GLP-2 release due to intact alpha-La and other alpha-La hydrolysates. Free aminoacid concentrations and molecular size distributions did not differ among alpha-La hydrolysates, including LaHPA. In rat pups reared with milk formulae containing alpha-La or LaHPA, LaHPA significantly promoted small intestinal elongation and increased the number of crypt epithelial cells compared with a formula containing intact alpha-La. LaHPA administration also increased the maltase:lactase activity ratio, a marker of maturation of the intestinal mucosa. In conclusion, milk proteins stimulate GLP-2 secretion and contribute to growth and maturation of the small intestine in suckling rats. J. Nutr. 139: 1322-1327, 2009.

Synbiotic promotion of epithelial proliferation by orally ingested encapsulated Bifidobacterium breve and raffinose in the small intestine of rats
Satoshi Ishizuka, Ami Iwama, Achmad Dinoto, Akarat Suksomcheep, Kohshi Maeta, Takanori Kasai, Hiroshi Hara, Atsushi Yokota
MOLECULAR NUTRITION & FOOD RESEARCH, 53, S62, S67, WILEY-V C H VERLAG GMBH, May 2009, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, We evaluated the effects of Bifidobacterium breve JCM1192(T) and/or raffinose on epithelial proliferation in the rat small and large intestines. WKAH/Hkm Slc rats (4 wk old) were fed a control diet, a diet supplemented with either encapsulated B. breve (30 g/kg diet, 1.5 x 10(7) colony-forming unit/g capsule) or raffinose (30 g/kg diet), or a diet supplemented with both encapsulated B. breve and raffinose, for 3 wk. Epithelial proliferation in the small intestine, as assessed by bromodeoxyuridine immunohistochemistry, was increased only in the B. breve plus raffinose-fed group. We determined the number of bifidobacteria in cecal contents using fluorescence in situ hybridization and confirmed the presence of ingested B. breve only in the B. breve plus raffinose-fed group. This suggests that the ingested B. breve cells used raffinose and were activated in the small intestine, where they subsequently influenced epithelial proliferation. In conclusion, we found a prominent synbiotic effect of encapsulated B. breve in combination with raffinose on epithelial proliferation in rat small intestine but not in large intestine. To our knowledge, this is the first report of a synbiotic that affects epithelial proliferation.

Depletion of CD8 alpha(+) lymphocytes attenuates CCL28 expression in villus epithelia in rats
Jae-Sung Lee, Saki Kamada, Yuri Takami, Kohsuke Oka, Yuichi Ochiai, Hitoshi Iwaya, Hiroshi Hara, Satoshi Ishizuka
IMMUNOLOGY LETTERS, 124, 1, 50, 54, ELSEVIER SCIENCE BV, May 2009, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, To examine the involvement of CD8 alpha(+) intraepithelial lymphocytes (IELs) in chemokine expression by villus epithelial cells, villus and crypt fractions were collected by mechanical isolation using a chelating buffer and specific antibodies in CD8 alpha(+) cell-depleted rats. A larger population of CD8 alpha(+) cells was observed by histochemical evaluation in villus epithelia than in crypt epithelia in rat small intestine, and CCL9 and CCL28 expression was higher in the crypt fraction than in the villus fraction. The mRNA expression of CCL28 in villus fractions isolated from rat small intestinal mucosa was significantly reduced compared to that of CCL9, and was accompanied by CD8 alpha depletion. Using a combined histochemical and flow cytometric approach, CD8 alpha alpha(+) cells were detected in the intraepithelial region of the villus epithelium. Thus, CCL28 expression in villus epithelial cells is partially supported by CD8 alpha alpha(+) cells, and CD8 alpha(+) IELs are involved in CCL28 expression. (c) 2009 Elsevier B.V. All rights reserved.

Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS
Masahito Hagio, Megumi Matsumoto, Michihiro Fukushima, Hiroshi Hara, Satoshi Ishizuka
JOURNAL OF LIPID RESEARCH, 50, 1, 173, 180, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jan. 2009, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, To evaluate bile acid (BA) metabolism in detail, we established a method for analyzing BA composition in various tissues and intestinal contents using ultra performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). Twenty-two individual BAs were determined simultaneously from extracts. We applied this method to define the differences in BA metabolism between two rat strains, WKAH and DA. The amount of total bile acids (TBAs) in the liver was significantly higher in WKAH than in DA rats. In contrast, TBA concentration in jejunal content, cecal content, colorectal content, and feces was higher in DA rats than in WKAH rats. Nearly all BAs in the liver were in the taurine-or glycine-conjugated form in DA rats, and the proportion of conjugated liver BAs was up to 75% in WKAH rats. Similar trends were observed for the conjugation rates in bile. The most abundant secondary BA in cecal content, colorectal content, and feces was hyodeoxycholic acid in WKAH rats and v-muricholic acid in DA rats. Analyzing detailed BA profiles, including conjugation status, in a single run is possible using UPLC/ESI-MS. This method will be useful for investigating the roles of BA metabolism under physiological and pathological conditions. Hagio, M., M. Matsumoto, M. Fukushima, H. Hara, and S. Ishizuka. Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS. J. Lipid Res. 2009. 50: 173-180.

Attenuation of interferon-gamma mRNA expression in activated Jurkat T cells by exogenous zinc via down-regulation of the calcium-independent PKC-AP-1 signaling pathway
Kumiko Hayashi, Satoshi Ishizuka, Chieko Yokoyama, Toshihisa Hatae
LIFE SCIENCES, 83, 1-2, 6, 11, PERGAMON-ELSEVIER SCIENCE LTD, Jul. 2008, [Peer-reviewed]
English, Scientific journal, Zinc is known to modulate a wide variety of cellular functions including anti-inflammatory responses. We examined the intracellular signaling pathways that contribute to the regulation of interferon-gamma (IFN-gamma) by zinc in activated human Jurkat T cells. Zinc significantly reduced IFN-gamma expression and activator protein-1 (AP-1) signaling in cells activated by phorbol 12-myristate 13-acetate (PMA) and phytohemagglutinin (PHA) without affecting cell viability. Moreover, partial inhibition of AP-1 activity by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, resulted in marked reduction of IFN-gamma transcription. We also found that this inhibitory effect of zinc on AP-1 signaling was abolished by treatment with rottlerin, a selective inhibitor of calcium-independent protein kinase C (PKC). These results suggest a novel target of zinc in the calcium-independent protein kinase C-AP-1 pathway to regulate endogenous IFN-gamma gene expression in activated T cells. (C) 2008 Elsevier Inc. All rights reserved.

Modulation of rat cecal microbiota by administration of raffinose and encapsulated Bifidobacterium breve
A Dinoto, A Suksomcheep, S Ishizuka, H Kimura, S Hanada, Y Kamagata, K Asano, F Tomita, A Yokota
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 72, 1, 784, 792, AMER SOC MICROBIOLOGY, Jan. 2006, [Peer-reviewed]
English, Scientific journal, To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192(T) cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Sic rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BID). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.

Promotive effects of non-digestible disaccharides on rat mineral absorption depend on the type of saccharide
P Asvarujanon, S Ishizuka, H Hara
NUTRITION, 21, 10, 1025, 1035, ELSEVIER SCIENCE INC, Oct. 2005, [Peer-reviewed]
English, Scientific journal, Objective: We examined the effects of feeding non-digestible disaccharides, difructose anhydride III (DFAIII), maltitol, melibiose and, cellobiose, on calcium, magnesium, and iron absorption in comparison with fructo-oligosaccharide (FOS) in normal and ovariectomized rats.
Methods: In experiment 1, six groups of male Sprague-Dawley rats were fed a control diet (100 g of cellulose/kg of diet), test diets containing 30 g of FOS, or the four non-digestible disaccharides in place of the cellulose in the control diet for 4 wk. In experiment 2, two groups of female Sprague-Dawley rats (sham or ovariectomized) were assigned to one of four subgroups and fed the control or test diet containing FOS, DFAIII, and melibiose for 5 wk. Feces and cecal contents were collected to evaluate mineral absorption and cecal fermentation.
Results: In experiment 1, calcium absorption in all the disaccharides groups except the cellobiose group, magnesium absorption in all test diet groups, and iron absorption in the FOS, DFAIII, and melibiose groups were higher than those in the control group. In ovariectomized rats (experiment 2), calcium absorption in the DFAIII and melibiose groups, magnesium absorption in all test diet groups, and iron absorption in the DFAIII group alone were higher than those in the control group. Cecal organic acids were positively and pH was negatively correlated with the absorption of these minerals, although the effects varied.
Conclusion: Non-digestible disaccharides increase calcium, magnesium, and iron absorption in normal and ovariectomized rats; however, the effects depend on the disaccharide tested, a fact that may be partly associated with the cecal fermentation of these disaccharides. (c) 2005 Elsevier Inc. All rights reserved.

Strain and age-related changes in the localization of intestinal CD161(+) natural killer cells and CD8(+) Intraepithelial lymphocytes along the longitudinal crypt axis in inbred rats
H Xu, S Imanishi, K Yamada, H Hara, S Ishizuka
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 69, 3, 567, 574, TAYLOR & FRANCIS LTD, Mar. 2005, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, Using a cell position approach, this study indicates that the frequency of CD161(+) natural killer (NK) cells in the epithelia of DA rats was greater than that of WKAH and F344 rats. We further divided the epithelia into proliferating and differentiated regions according to the localization of BrdU-incorporated cells. Comparison between the different regions indicates that a majority of CD161(+) NK cells were located in the proliferating region. With age, a decline in the number of CD161(+) NK cells and CD8(+) intraepithelial lymphocytes (IELs) was observed in the distal colon, especially in the proliferating region of all three strains. Taken together with our previous report that DA rats have far stronger resistance in the colon to preneoplastic lesion than do other strains, these results indicate that CD161(+) NK cells play an important role in immune-surveillance at the bottom of the crypt.

Fermentable dietary fiber potentiates the localization of immune cells in the rat large intestinal crypts
S Ishizuka, S Tanaka, H Xu, H Hara
EXPERIMENTAL BIOLOGY AND MEDICINE, 229, 9, 876, 884, SOC EXPERIMENTAL BIOLOGY MEDICINE, Oct. 2004, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, Intestinal crypts are composed of a well-defined hierarchy of epithelial cells, and proliferating epithelial cells reside close to the bottom of the crypts-even in the large intestine. We investigated whether CD8(+) and CD4(+) intraepithelial lymphocytes (IELs) and CD161(+) natural killer (NK) cells localized in proliferating or differentiated epithelial region of cecum and colon. Both proliferating epithelial layer cells and the immune cells along the longitudinal crypt axis of the large intestine were measured histochemically. Dietary intervention revealed that the physiological localization of the immune cells in the longitudinal crypt axis depended on the immune cell type. CD8(+) IELs were preferentially located among differentiated epithelial cells. In contrast, CD161(+) NK cells were located adjacent to the epithelial cells at the bottom of crypt. Cecal crypts contained significantly larger numbers of CD8(+) IELs than did colonic crypts. However, there was only a minor population of CD4(+) IEL in the cecal and colonic epithelia. Some dietary fibers increased the densities of CD8(+) IELs and CD161(+) NK cells in the cecum, with the magnitude of response varying among the types of fiber. There was a significant relationship between SCFA and the localization of immune cells, especially CD8(+) IEL and CD161(+) NK cells, which are considered to be involved in the maintenance of epithelial homeostasis.

Inhibitory effects of psyllium on rat mineral absorption were abolished by reduction of viscosity with partial hydrolysis
P Asvarujanon, S Ishizuka, H Hara
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 68, 8, 1737, 1742, TAYLOR & FRANCIS LTD, Aug. 2004, [Peer-reviewed]
English, Scientific journal, Psyllium husk, a highly viscous fiber, has beneficial effects for health, but has been reported to inhibit absorption of calcium. The present study found the effects of fiber viscosity on calcium, magnesium, and zinc absorption with partially degraded psyllium preparations to be one fifth viscosity (LD-HP) and one seventieth viscosity (HD-HP) using normal and ovariectomized rats. Magnesium absorption was reduced with ingestion of intact psyllium (50 g/kg diet) for 4 weeks but this reduced absorption was increased with lower viscous psyllium preparations. Moreover, the absorption in the HD-HP group was higher than in the control group (100 g cellulose/kg diet) in ovariectomized rats. Changes in calcium and zinc absorption were similar to those in magnesium absorption. Cecal pH was reduced only in rats fed HD-HP in both normal and ovariectomized rats. These results indicate that reduction of psyllium viscosity tends to counter inhibitory effects on mineral absorption.

Reducing effect of ingesting tannic acid on the absorption of iron, but not of zinc, copper and manganese by rats
K Afsana, K Shiga, S Ishizuka, H Hara
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 68, 3, 584, 592, TAYLOR & FRANCIS LTD, Mar. 2004, [Peer-reviewed]
English, Scientific journal, Interest in the beneficial effects of polyphenols, including tannic acid (TA), is increasing, although, these compounds also have adverse effects; for example, on the absorption of iron (Fe), and possibly other trace minerals. We examined the effect of a graded dose of TA on the absorption of Fe and compared with that of zinc (Zn), copper (Cu) and manganese (Mn) in rats. We also investigated the effect of TA on cecal fermentation which plays a role in absorption. In Experiment 1, to set the optimum dose of Fe, male Sprague-Dawley rats (weighing 70-90 g) after acclimatization were fed with different levels of dietary Fe (5, 10, 20, 30 and 35 mg/ kg). We observed that the hematocrit (Ht), serum Fe concentration and transferrin saturation (%) were each reduced in those rats fed less than 20 mg/kg Fe in a dose-dependant manner. In Experiment 2, the rats were fed with test diets containing the minimum required level of Fe, 30 mg/kg diet, with (5, 10, 15 and 20 g/kg diet) or without TA for a period of three weeks. Feeding a diet containing more than 10 g TA/kg diet, but not 5 g TA/kg diet, reduced the hemoglobin concentration (Hb), Ht and serum Fe concentration due to decreased Fe absorption. In contrast, the Zn, Cu and Mn absorption was not affected by TA feeding. It is also demonstrated that liver Fe, but not the Zn, Cu and Mn contents, were lower in the TA groups than in the TA-free control group. Feeding TA slightly decreased the pH value of the cecal contents with an increase in the major short-chain fatty acid pool. About 15% of the ingested TA were recovered in the feces of each TA-fed group. Our results demonstrate that more than 10 g TA/kg diet induced anemia by reducing the Fe absorption, although there was no effect on the absorption of other important trace-minerals. Our findings suggest that the usual intake of polyphenols is relatively safe, but that a high intake by supplementation or by dietary habit of tannin affects only the Fe level.

Intrarectal administration of deoxycholate selectively reduces small aberrant crypt foci in the rat colorectum
S Ishizuka, H Sakai, Y Yoshioka
NUTRITION RESEARCH, 23, 12, 1703, 1708, PERGAMON-ELSEVIER SCIENCE LTD, Dec. 2003, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, We investigated whether intrarectal application of deoxycholate (DCA), taurine, DCA with taurine or taurodeoxycholate modifies the formation of aberrant crypts (ACs) induced by N,N'-dimethylhydrazine in the colorectum of WKAH rats. Administration of DCA led to a significant reduction in the number of aberrant crypt foci (ACF) in the colorectum. Interestingly, the administration of DCA selectively decreased the number of ACF with 1-3 aberrant crypts/focus (AC/focus) more effectively than did that of saline or taurine. As a result, there was an increasein the AC/focus ratio in the WKAH rats administered DCA. In conclusion, increased luminal concentration of DCA with enema resulted in a significant reduction in smaller ACF, which raised the AC/focus ratio in the colorectum of WKAH rats. (C) 2003 Elsevier Inc. All rights reserved.

Poly(ADP-ribose) polymerase-1 is a survival factor for radiation-exposed intestinal epithelial stem cells in vivo
S Ishizuka, K Martin, C Booth, CS Potten, G de Murcia, A Burkle, TBL Kirkwood
NUCLEIC ACIDS RESEARCH, 31, 21, 6198, 6205, OXFORD UNIV PRESS, Nov. 2003, [Peer-reviewed], [Lead author], ［Internationally co-authored］
English, Scientific journal, Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme mediating the cellular response to DNA strand breaks. It plays a critical role in genomic stability and survival of proliferating cells in culture undergoing DNA damage. Intestinal epithelium is the most proliferative tissue in the mammalian body and its stem cells show extreme sensitivity to low-level genotoxic stress. We investigated the role of PARP-1 in the in vivo damage response of intestinal stem cells in crypts of PARP-1(-/-) and control mice following whole-body gamma-irradiation (1 Gy). In the PARP-1(-/-) mice there was a significant delay during the first 6 h in the transient p53 accumulation in stem cells whereas an increased number of cells were positive for p21(CIP1/WAF1). Either no or only marginal differences were noted in MDM2 expression, apoptosis, induction of or recovery from mitotic blockage, or inhibition of DNA synthesis. We further observed a dose-dependent reduction in crypt survival measured at 4 days post-irradiation in control mice, and this crypt-killing effect was significantly potentiated in PARP-1(-/-) mice. Our results thus establish that PARP-1 acts as a survival factor for intestinal stem cells in vivo and suggest a functional link with early p53 and p21(CIP1/WAF1) responses.

Ingestion of an indigestible saccharide, difructose anhydride III, partially prevents the tannic acid-induced suppression of iron absorption in rats
K Afsana, K Shiga, S Ishizuka, H Hara
JOURNAL OF NUTRITION, 133, 11, 3553, 3560, AMER INST NUTRITION, Nov. 2003, [Peer-reviewed]
English, Scientific journal, Dietary tannic acid (TA) inhibits iron absorption and some indigestible oligosaccharides have been shown to promote mineral absorption. In this study, we examined whether difructose anhydride III (DFA III) or fructooligosaccharide (FOS) stimulate iron absorption in TA-fed rats. Two experiments were conducted using male Sprague-Dawley rats (weighing 90-110 g) in a randomized block design. Rats were fed control, DFA III or FOS (30 g/kg) diets in expt. 1, and control, TAcontrol, TAFOS or TADFA III (TA, 15 g/kg) diets in expt. 2 for 3 wk during which blood sampling was performed weekly and fecal collection twice. In expt. 1, apparent iron absorption was higher (P < 0.001) in the DFA III-fed (65.7 and 55.9%, d 8-10 and 19-21) and FOS-fed (59.9%, d 19-21) groups than in the control group (48.4 and 45.4%, d 8-10 and 19-21) without differences in blood hemoglobin concentrations or hematocrits. TA feeding reduced hemoglobin concentrations and hematocrits (119.1 g/L, 0.360; P < 0.001), and the feeding of TADFA III partially improved this anemic condition (129.6 g/L, 0.403), whereas TAFOS feeding did not influence these variables (120.6 g/L, 0.342; expt. 2). Iron absorption was lower in the TA-fed groups (19.8%; P < 0.001) than in the control group (49.4%), whereas the absorption in both TA-fed indigestible sugar groups was higher (DFA III, 43.2 and 38.2%, d 8-10 and 19-21; FOS, 39.4%, d 8-10; P < 0.001) than in the TA-control group except for the TAFOS-fed group (25.1%, d 19-21). Serum iron concentrations, unsaturated iron-binding capacities, total iron-binding capacities and transferrin saturations (%) were not improved by the feeding of TADFA III or TAFOS. Furthermore, liver iron concentrations were decreased by TA feeding (P < 0.001) and were not increased by the feeding of indigestible sugars. The feeding of DFA III or FOS decreased the pH of the cecal contents (P < 0.001) while increasing major organic acid pools. In all groups fed TA, similar to18% of the ingested TA was recovered in the feces. Our results demonstrate that TA reduces iron absorption and induces anemia, conditions that are partially prevented by the feeding of DFA III, but not FOS.

Relationship between formation of aberrant crypts and acute responses of colonic epithelial cells to genotoxic treatment among inbred rat strains
S Ishizuka, A Takeuchi, M Hagio, M Mohara, H Sakai, K Yamada
CANCER LETTERS, 196, 2, 135, 141, ELSEVIER SCI IRELAND LTD, Jul. 2003, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, DNA damage such as chemical carcinogen or gamma-rays induces aberrant crypts in the rat colorectum. We demonstrated that formation of aberrant crypts is different among inbred rat strains (WKAH, DA and F344/N). DA had less preneoplastic lesions in the colorectum than the others regardless of the way of DNA damage. We analyzed changes in in vivo number of colonic epithelial cells undergo mitosis, DNA synthesis and apoptosis following DNA damage histochemically. It is indicated that rapid onset of G(1) arrest and termination of G(2)/M arrest and apoptosis in damaged epithelial cells is important to reduce subsequent formation of the preneoplastic lesions. (C) 2003 Elsevier Science Ltd. All rights reserved.

Reduction of aberrant crypt foci by ingestion of polydextrose in the rat colorectum
S Ishizuka, T Nagai, H Hara
NUTRITION RESEARCH, 23, 1, 117, 122, PERGAMON-ELSEVIER SCIENCE LTD, Jan. 2003, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, To investigate the effect of dietary polydextrose on development of aberrant crypt foci (ACF), we fed rats with polydextrose-containing diet in various timing before or after 1,2-dimethythydrazine (DMH) injection and measured ACF parameters. Every parameter for ACF was significantly reduced in the rats fed polydextrose from day 7 before DMH injection than in the fiber-free diet fed rats. This study shows that ingestion of polydextrose has protective effect on ACF especially in the rectum and the timing of dietary treatment is critical for inhibition of ACF development. (C) 2003 Elsevier Science Inc. All rights reserved.

Liberation of free fatty acids from milk fat by lactic acid bacteria
A Kimura, H Kumura, S Ishizuka, K Mikawa, K Shimazaki, Z Saito
MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL, 58, 11-12, 609, 611, A V A AGRARVERLAG, 2003, [Peer-reviewed]
English, Scientific journal, The lipolytic activity of Lactococcus lactis ssp. cremoris was analyzed using gas chromatography and an agar plate method. A cell free extract obtained by sonication of the living cells was inactive toward milk fat triglycerides. However, production of free fatty acids by the cell free extract was detected when the substrate had been pretreated with indigenous bovine milk lipase. In the cell free extract, monoglyceride lipase activity was detected, independent of the number of carbons in the fatty acids. Thus, it was concluded that the bovine milk lipase degraded milk fat triglycerides to monoglycerides, which were then ready to be hydrolyzed by a monoglyceride lipase in the cell free extract to release free fatty acids. It is likely that the lipolytic contribution of this bacterial strain contributes to flavour development during cheese ripening, due mainly to the hydrolytic activity toward monoglycerides in cheese curd.

Modulation of CD8(+) intraepithelial lymphocyte distribution by dietary fiber in the rat large intestine
S Ishizuka, S Tanaka
EXPERIMENTAL BIOLOGY AND MEDICINE, 227, 11, 1017, 1021, SOC EXPERIMENTAL BIOLOGY MEDICINE, Dec. 2002, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, We studied whether ingestion of dietary fiber modifies the distribution of intraepithelial lymphocytes (IIEL) in a physiological condition. Male WKAH rats were fed diets either with fiber (sugar beet fiber or crystalline cellulose, 100 g/kg diet each) or without fiber for 3 weeks. The number of CD8(+), CD4(+), and NKR-P1(+) IIEL per epithelial layer in the crypt section of the cecum, proximal colon, and distal colon were scored by immunohistochemical staining. We found that the proportion of CD8(+) IEL was greater in the cecal mucosa and was gradually reduced toward the distal large intestine in general. In contrast, there was no difference in the proportion of CD4(+) and NKR-P1(+) IEL in the large intestine. Dietary sugar beet fiber, but not crystalline cellulose, increased the proportion of CD8(+) IEL, especially in the cecal mucosa, but not the CD4(+) and NKR-P1(+) IEL. Analysis of cecal organic acid concentration confirmed higher concentrations of acetate and butyrate, and lower concentration of succinate and isovalerate, in the cecum of the rats fed sugar beet fiber than other diets. These results indicate that ingestion of some dietary fiber modulates local cell proliferation of a progenitor of CD8(+) IEL or promotes homing of CD8(+) T cells into the large intestinal epithelium, most likely via the fermentation in the luminal contents.

Dietary sugar beet fiber ameliorates diarrhea as an acute gamma-radiation injury in rats
S Ishizuka, S Ito, T Kasai, H Hara
RADIATION RESEARCH, 154, 3, 261, 267, RADIATION RESEARCH SOC, Sep. 2000, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, Gamma radiation induces diarrhea as an acute injury, We have studied whether ingestion of sugar beet fiber influences radiation-induced diarrhea, Abdominal irradiation with gamma rays induced diarrhea in male Wistar/ST rats from 2 to 7 days after a single sublethal dose. The body weight of the irradiated rats was decreased temporarily at 4 days after irradiation regardless of the ingestion of sugar beet fiber, At day 8, it returned to almost the same level as that of unirradiated rats. A change in daily food intake resulted in a pattern similar to that for body weight. Dietary sugar beet fiber had little significant effect on the changes in body weight and daily food intake, and its ingestion significantly decreased gamma-ray-induced diarrhea, Changes in biochemical and histological parameters in intestinal mucosa (small intestine, cecum and colon) were not greatly influenced by the ingestion of sugar beet fiber through the periods of diarrhea, It was concluded that dietary sugar beet fiber ameliorated the diarrhea induced by abdominal irradiation. We suggest that the inhibitory effect of the ingestion of sugar beet fiber Is due to its effects on the luminal environment, such as support for bacterial function in the luminal contents in the colon of animals that ingest sugar beet fiber, (C) 2000 by Radiation Research Society.

Dietary sugar beet fiber prevents the increase in aberrant crypt foci induced by gamma-irradiation in the colorectum of rats treated with an immunosuppressant
T Nagai, S Ishizuka, H Hara, Y Aoyama
JOURNAL OF NUTRITION, 130, 7, 1682, 1687, AMER INST NUTRITION, Jul. 2000, [Peer-reviewed], [Corresponding author]
English, Scientific journal, We demonstrated recently that gamma-irradiation can induce aberrant crypt fool (ACF) in the rat colorectum, The aim of this study was to evaluate the effect of dietary sugar beet fiber (SBF) on the distribution of the CD8(+) intraepithelial lymphocyte (IEL) in the colorectum and on the number of gamma-irradiation-induced ACF of rats administered anti-asialo GM1 (alpha AGM1) as an immunosuppressant. Wistar/ST rats fed a fiber-free diet or the diet supplemented with SBF (100 g/kg diet) were administrated alpha AGM1 or normal rabbit serum as a control during the initiation period with gamma-irradiation. At 5 and 9 wk after the first irradiation, ACF and total aberrant crypts (AC) per area in the colorectum were counted. The numbers of ACF (P = 0.0010) and AC (P = 0.0635) per unit area were lower in the SBF-fed group than in the rats fed the fiber-free diet. alpha AGM1 administration significantly raised the number of ACF (P = 0.0001) and AC (P = 0.0006) per area in the colorectum. Moreover, alpha AGM1 administration during the initiation period reduced the number of CD8(+) IEL per 100 cells in the epithelial layer (P = 0.0001) of the colon. These results demonstrate that reduction of the number of CD8(+) IEL per 100 cells in the epithelial layer as a result of alpha AGM1 administration promotes the formation of irradiation-induced ACF in the colorectum. The number of CD8(+) IEL per 100 cells in epithelial layer was lower in the group fed the fiber-free diet than in the SBF-fed group (P = 0.0522). These results indicated that the ingestion of dietary SBF suppressed gamma-irradiation-induced ACF formation through the immune surveillance in the colorectal mucosa.

Ingestion of sugar beet fiber enhances irradiation-induced aberrant crypt foci in the rat colon under an apoptosis-suppressed condition
S Ishizuka, S Ito, M Onuma, T Kasai, K Aoyama, H Hara
CARCINOGENESIS, 20, 6, 1005, 1009, OXFORD UNIV PRESS, Jun. 1999, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, The induction of aberrant crypt foci (ACF) by irradiation of gamma-rays (Co-60), and the effect of dietary sugar beet fiber (SBF) on irradiation-induced ACF were examined. We found that abdominal irradiation of gamma-rays could induce ACF in the rat colon. The irradiation was performed once a week at a dose rate of 2 or 3 Gy per irradiation. Irradiation-induced ACF were observed in the colon at 10 weeks after the first irradiation at dose of 2 Gy for six times or 3 Gy for four times. Dietary SBF had no effect on the number of ACF, aberrant crypts (AC) or AC/focus induced by abdominal gamma-irradiation. However, an ingestion of SBF resulted in an increase in the number of these parameters in apoptosis-suppressed rats by cycloheximide (CHX). An injection of CHX suppressed irradiation-induced apoptosis of the colonic epithelial cells for at least 6 h after the irradiation. In CHX-injected rats, an ingestion of SBF significantly increased the number of ACF, AC and AC/focus compared with fiber-free fed rats at 9 weeks after the first irradiation. On the other hand, in saline-injected rats, no significant difference was found between SBF and fiber-free diets in the number of ACF, AC and AC/focus through the experimental period. These results suggest that dietary SBF may be involved in the elimination of abnormal cells from an irradiated colon through the apoptosis of colonic epithelial cells, In this study, we have shown a new method for inducing ACF by using gamma-rays which were not influenced by luminal contents such as bacterial enzyme, at least in the initiation stage.

Dietary wheat bran modulates proliferating cell nuclear antigen-labeling index of the rat colorectum after treatment with 1,2-dimethylhydrazine
S Ishizuka, T Kasai
JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY, 43, 6, 665, 671, CENTER ACADEMIC PUBL JAPAN, Dec. 1997, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, We investigated whether dietary wheat bran (Wb) influences the proliferating cell nuclear antigen (PCNA)-labeling index (LI) of the colorectum of rats after the injection of 1,2-dimethylhydrazine (DMH). Male Wistar/ST rats were divided into four groups and given either a fiber-free diet or diets supplemented with Wb at the expense of the whole diet (5, 10 or 20 g/100 g diet). Then, they were subcutaneously injected with a single dose of DMH (20 mg/kg body weight) or the vehicle. At four weeks after the treatment, frozen sections of the colorectum were immunostained with anti-PCNA antibody (19F4). In the fiber-free group, DMH treatment significantly increased the PCNA-LI of the distal colon and rectum. Among the DMH treatment groups, the PCNA-LI of the distal colon in the Wb 5 g/100 g diet group was significantly lower than that in the fiber-free group. The PCNA-LI of the distal colon tended to increase as the amount of Wb supplemented was increased in the DMH-treated groups except for the fiber-free group. A similar trend was observed for the rectum. In conclusion, the ingestion of Wb diminished the increase in PCNA-LI in rat colorectum induced by DMH injection.

Suppression of the number of aberrant crypt foci of rat colorectum by ingestion of sugar beet fiber regardless of administration of anti-asialo GM(1)
S Ishizuka, T Kasai
CANCER LETTERS, 121, 1, 39, 43, ELSEVIER SCI IRELAND LTD, Dec. 1997, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, Anti-asialo GM(1) serum (AGM1) reduces natural killer (NK) activity in vitro and in vivo. We investigated the effect of ingestion of sugar beet fiber (SBF) on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and whether the effect was maintained under NK-reducing conditions by AGM1 injection. The ingestion of SBF decreased the number of ACF in the colorectum at 4 weeks after treatment with DMH. Dietary SBF had a suppressive effect on the formation of ACF regardless of the administration of AGM1. These results suggest that the suppressive effect created by the ingestion of SBF may overwhelm the effect of AGM1 treatment on ACF formation. (C) 1997 Elsevier Science Ireland Ltd.

Changes in the number and apoptosis of epithelial cells in the colorectum of wheat bran-fed rats soon after administering 1,2-dimethylhydrazine
S Ishizuka, K Sonoyama, T Kasai
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 61, 8, 1337, 1341, TAYLOR & FRANCIS LTD, Aug. 1997, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, We investigated the effect of dietary wheat bran (Wb) on colonic tumorigenesis soon after a single administration of 1,2-dimethylhydrazine (DMH). Rats that had been fed on either a fiber-free diet or a 20% Wb diet were injected with 1,2-dimethylhydrazine (20 mg/kg body weight). At 6 h, 12 h, 1 d, 3 d, or 7 d after the injection, the colorectum was excised for histological analyses, The number of crypt cells more rapidly recovered in the 20% Wb group than in the fiber-free group after its temporary reduction by injection of DMH. At 6h after the DMH treatment, the apoptotic cells were significantly greater in number in the fiber-free group than in the 20% Wb group, In contrast, those in distal colon were significantly fewer in the fiber-free group than in the 20% Wb group at 7 d after the treatment, These results suggest that the ingestion of Wb affected the turnover of colonic epithelial cells and would thereby bring about a protective effect against DMH-induced tumorigenesis.

Inhibitory effect of dietary wheat bran on formation of aberrant crypt foci in rat colon induced by a single injection of 1,2-dimethylhydrazine
S Ishizuka, T Kasai
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 60, 12, 2084, 2085, TAYLOR & FRANCIS LTD, Dec. 1996, [Lead author, Corresponding author]
English, Scientific journal, The frequency of appearance of aberrant crypt foci (ACF) in the distal colon was significantly lower in rats fed a high fiber (20% wheat bran) diet than in those fed a fiber-free one at 4 weeks after a single injection of 1,2-dimethylhydrazine (DMH, 20 mg/kg), although crypt/ACF was high in the former relative to the latter, This result suggests that dietary wheat bran effectively serves as a regulator of ACF frequency at early stages after DMH injection.

Administration of anti-asialo GM(1) serum increases aberrant crypt foci induced by 1,2-dimethylhydrazine in the large bowel of rats
S Ishizuka, T Nagai, T Kasai
JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY, 42, 6, 603, 608, CENTER ACADEMIC PUBL JAPAN, Dec. 1996, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, We investigated the influence of the administration of anti-asialo GM(1) antibody on aberrant crypt foci (ACF) formation induced by a single injection of 1,2-dimethylhydrazine (DMH). At four weeks after the injection of DMH (20 mg/kg body weight), the number of ACF and aberrant crypts were counted. Most ACF appeared in the distal large bowel, accounting for approximately 60% of the total ACF in both groups. Rats administered anti-asialo GM(1) had significantly more ACF in the distal colon, the rectum and the total large bowel as compared to control rats. A similar tendency was observed for the number of aberrant crypts. The increased number of ACF resulting from the administration of anti-asialo GM(1) was not accompanied by the enlargement of ACF size in every part of the colon. This study demonstrated that the administration of anti-asialo GM(1) at the initiation stage leads to an increase in ACF as well as aberrant crypts in the distal colon, rectum and total large bowel probably via the suppression of natural killer cells.

FOOD-DEPRIVATION INCREASES APOPTOTIC CELL COUNTS INDUCED BY 1,2-DIMETHYLHYDRAZINE IN RAT DESCENDING COLONIC AND RECTAL CRYPTS
S ISHIZUKA, K SONOYAMA, R NIKI
JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY, 40, 4, 363, 369, CENTER ACADEMIC PUBL JAPAN, Aug. 1994, [Peer-reviewed], [Lead author, Corresponding author]
English, The frequency of apoptosis after treatment with 1,2-dimethylhydrazine (DMH) was counted in the descending colonic and rectal crypts of food-deprived and fed rats. Food-deprived or fed rats were subcutaneously injected with DMH (100 mg/kg body weight). Six hours after the injection, apoptotic cells were observed in crypt regions by light microscopy. The incidence of DMH-induced apoptosis in food-deprived rats was significantly higher than in fed rats. The incidence appeared to be higher in descending colon than in rectum. PAS staining revealed that DMH treatment lowered mucin secretion in crypts, which was substantially lowered by food deprivation. The effect of food deprivation on apoptosis induced by DMH may be due to the decrease in mucus barrier against DMH.

Influence of homogenization of milk on fatty acid contents in cheese
Ishizuka Satoshi, Saito Zenichi
Memoirs of the Faculty of Agriculture, Hokkaido University, 19, 2, 289, 294, Hokkaido University, 1994, [Peer-reviewed], [Lead author]

Basic Protocols in Foods and Nutrition　　　　　　　　　　　　　　　
Shota Hori, Hongxia Liu, Riho Yamada, Shun Ichitsubo, Ayana Sakaguchi, Fumika Yokoyama, Satoshi Ishizuka, Extraction of Bile Acids from Biological Samples and Quantification Using Ultra-High-Performance Liquid Chromatography-Orbitrap Mass Spectrometry
Humana Press, Springer, 2022, [Contributor]

Metabolic Profiling, Methods in Molecular Biology　　　　　　　　　　　　　　　
Hagio M, Matsumoto M, Ishizuka S, Bile acid analysis in various biological samples using ultra performance liquid chromatography/electrospray mass spectrometry (UPLC/ESI-MS)
Humana Press, Springer, 2011, [Contributor]

消化管の栄養・生理と腸内細菌　　　　　　　　　　　　　　　
石塚 敏, 腸管免疫系細胞の存在位置とその粘膜恒常性維持における役割
Hindgut Club Japan, 2010, [Contributor]

Metabolic alteration induced by 12-hydroxylated bile acids and disease onsets　　　　　　　　　　　　　　　
Satoshi Ishizuka
第78回 日本栄養・食糧学会大会, 25 May 2024, Japanese, Nominated symposium
[Invited], [Domestic Conference]

12水酸化一次胆汁酸による回腸透過性の亢進作用　　　　　　　　　　　　　　　
石塚 敏
第77回 日本栄養・食糧学会大会, 14 May 2023
[Domestic Conference]

12α水酸化一次胆汁酸による消化管バリア機能の脆弱化　　　　　　　　　　　　　　　
石塚 敏
15 Jul. 2022, 細胞工学研究会, Japanese, Invited oral presentation
Japan, [Invited], [Domestic Conference]

胆汁酸代謝に着目した未病モデルの構築　　　　　　　　　　　　　　　
石塚 敏
第2回 ファーマラボEXPO大阪 アカデミックフォーラム, 11 Mar. 2022, Public discourse

12α-Hydroxylated bile acid as a biomarker in an early phase of noncommunicable diseases　　　　　　　　　　　　　　　
Satoshi Ishizuka
Hokkaido University-University of Melbourne Virtual Conference on Healthy Ageing, 08 Mar. 2022, English, Nominated symposium
[International presentation]

Diet-related presymptomatic disease models in experimental animals　　　　　　　　　　　　　　　
石塚 敏
第3回 ファーマラボEXPO東京 アカデミックフォーラム, 09 Dec. 2021, Japanese, Public discourse

ラットにおける12α水酸化胆汁酸による肝脂質蓄積と関連症状　　　　　　　　　　　　　　　
石塚 敏
第75回 日本栄養・食糧学会大会, 04 Jul. 2021, Japanese, Nominated symposium
03 Jul. 2021 - 04 Jul. 2021, [Invited]

高脂肪食摂取による代謝変動と12α水酸化胆汁酸の分泌増大　　　　　　　　　　　　　　　
石塚 敏
日本農芸化学会 2021年度仙台大会, 19 Mar. 2021, Japanese, Nominated symposium
18 Mar. 2021 - 19 Mar. 2021, [Invited]

腸内環境を介する食の作用 機能性食品の作用評価事例　　　　　　　　　　　　　　　
石塚 敏
日本薬局協励会 令和元年度学術繁栄講座, 16 Oct. 2019, Japanese, Public discourse
[Invited], [Domestic Conference]

未病としての軽度亜鉛欠乏　　　　　　　　　　　　　　　
石塚 敏
第８回 愛媛大学食品健康科学研究セミナー, 12 Sep. 2019, Japanese, Nominated symposium
[Invited], [Domestic Conference]

肝脂質蓄積におけるオリゴ糖の関与　　　　　　　　　　　　　　　
石塚 敏
食成分・栄養研究会セミナー・第367回 細胞工学研究会講演会, 29 Jun. 2019, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

実験動物で未病状態を再現する　　　　　　　　　　　　　　　
石塚 敏
食成分・栄養研究会セミナー, 04 Oct. 2018, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

糖代謝と胆汁酸　　　　　　　　　　　　　　　
石塚 敏
第7回 愛媛大学食品健康科学研究セミナー, 15 Sep. 2018, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

胆汁酸とミトコンドリア　　　　　　　　　　　　　　　
石塚 敏
7 Seas Project Research Meeting, 25 Aug. 2018, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

糖代謝変動における胆汁酸の寄与　　　　　　　　　　　　　　　
石塚 敏
第８回 オルソオルガノジェネシス検討会, 06 Jul. 2018, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

高脂肪食による胆汁酸代謝変動と耐糖能異常　　　　　　　　　　　　　　　
石塚 敏
第361回 細胞工学研究会講演会, 23 Jun. 2018, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

胆汁酸代謝から未病に迫る　　　　　　　　　　　　　　　
石塚 敏
第358回 細胞工学研究会講演会, 16 Dec. 2017, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

生体試料からの胆汁酸抽出・分析とその応用例　　　　　　　　　　　　　　　
石塚 敏
7 Seas Projectプレミーティング, 24 Feb. 2017, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

胆汁酸の代謝解析と関連病態　　　　　　　　　　　　　　　
石塚 敏
7 Seas Projectプレキックオフミーティング, 13 Oct. 2016, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

胆汁酸代謝に着目した新規病態モデルの確立　　　　　　　　　　　　　　　
石塚 敏
第5回 愛媛大学食品健康科学研究セミナー, 30 Sep. 2016, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

胆汁酸代謝と生活習慣病　　　　　　　　　　　　　　　
石塚 敏
分子細胞生物学セミナー, 12 Dec. 2015, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

胆汁酸代謝と消化管関連病態　　　　　　　　　　　　　　　
石塚 敏
第3回愛媛大学食品健康科学研究セミナー, 11 Sep. 2015, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

Metabolism of bile salts and intestinal microbiota　　　　　　　　　　　　　　　
ISHIZUKA Satoshi
Japanese Society of Gastroenterology – Asian-Pacific Association of Gastroenterology joint Conference, The 5th Asian Pacific Topic Conference., 22 Oct. 2014, English, Invited oral presentation
[Invited], [International presentation]

胆汁酸分子種の多様性と代謝性疾患における役割　　　　　　　　　　　　　　　
石塚 敏
第68回日本栄養・食糧学会大会, 01 Jun. 2014, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

Pathological aspects of bile acids: its metabolism and possible roles in metabolic syndrome.　　　　　　　　　　　　　　　
ISHIZUKA Satoshi
16th Seoul National University Hokkaido University joint symposium., 13 Dec. 2013, English, Invited oral presentation
[Invited], [International presentation]

Zinc as a key factor in experimental colitis and cytokine productions in isolated mesenteric leukocytes in rats.　　　　　　　　　　　　　　　
International Society for Nutraceuticals and Functional Foods, Nov. 2011, English, Public symposium
[Invited], [International presentation]

Dietary sugar beet fiber promotes an accumulation of CD8+ intraepithelial lymphocytes in small intestine in WKAH rats.　　　　　　　　　　　　　　　
9th Vahouny dietary fiber symposium, Jun. 2010, English, Public symposium
[International presentation]

胆汁酸の動態と消化管上皮に及ぼす作用　　　　　　　　　　　　　　　
第62回日本栄養・食糧学会大会関連学術集会 Hindgut Club Japanサテライトミーティング, May 2008, Japanese, Nominated symposium
[Invited], [Domestic Conference]

ラット腸粘膜免疫系細胞の食物繊維による局在調節と免疫監視能　　　　　　　　　　　　　　　
第4回日本食品免疫学会, May 2008, Japanese, Nominated symposium
[Invited], [Domestic Conference]

コール酸摂取による腸上皮細胞生存及び増殖の亢進　　　　　　　　　　　　　　　
第13回Hindgut Club Japanミーティング, Dec. 2007, Japanese, Nominated symposium
[Invited], [Domestic Conference]

胆汁酸による腸上皮の生存促進作用　　　　　　　　　　　　　　　
第61回日本栄養・食糧学会大会関連学術集会 Hindgut Club Japanサテライトミーティング, May 2007, Japanese, Nominated symposium
[Invited], [Domestic Conference]

Dietary fiber: a new modulator for distribution of immune cells around intestinal crypt.　　　　　　　　　　　　　　　
8th Vahouny Symposium, Sep. 2004, English, Nominated symposium
[Invited], [International presentation]

栄養学的観点から眺めた粘膜免疫に関する一つのアプローチ　　　　　　　　　　　　　　　
第57回 日本栄養・食糧学会大会 Hindgut Club Japanサテライトミーティング, May 2003, Japanese, Nominated symposium
[Invited], [Domestic Conference]

消化管上皮の恒常性維持に関する基礎的研究　　　　　　　　　　　　　　　
第57回 日本栄養・食糧学会大会, May 2003, Japanese, Public symposium
[Invited], [Domestic Conference]

消化管上皮の増殖と恒常性維持　　　　　　　　　　　　　　　
第８回Hindgut Club Japanミーティング, Dec. 2002, Japanese, Nominated symposium
[Invited], [Domestic Conference]

食品栄養学演習　　　　　　　　　　　　　　　
北海道大学大学院農学院

食品栄養学特論　　　　　　　　　　　　　　　
北海道大学大学院農学院

食資源科学総論　　　　　　　　　　　　　　　
北海道大学大学院農学院

生物機能化学実験IV　　　　　　　　　　　　　　　
北海道大学農学部

栄養化学I　　　　　　　　　　　　　　　
北海道大学農学部

食資源科学演習　　　　　　　　　　　　　　　
北海道大学大学院農学院

一般教育演習 食べ物の安全と安心　　　　　　　　　　　　　　　
北海道大学

食品栄養化学概論　　　　　　　　　　　　　　　
北海道大学農学部

食品科学特論演習　　　　　　　　　　　　　　　
北海道大学大学院農学院

食品科学特論　　　　　　　　　　　　　　　
北海道大学大学院農学院

2022 - Present
日本食品分析学会　　　　　　　　　　　　　　　

2012 - Present
American Society for Nutrition　　　　　　　　　　　　　　　

2005 - Present
The Molecular Biology Society of Japan　　　　　　　　　　　　　　　

2000 - Present
Hindgut Club Japan　　　　　　　　　　　　　　　

1995 - Present
Japan Society of Nutrition and Food Science　　　　　　　　　　　　　　　

1992 - Present
Japan Society for Bioscience, Biotechnology, and Agrochemistry　　　　　　　　　　　　　　　

12α水酸化胆汁酸による肝脂質蓄積機構ー脂質輸送と鉄代謝の寄与ー
科学研究費助成事業
Apr. 2023 - Mar. 2027
石塚 敏
日本学術振興会, 基盤研究(B), 北海道大学, 23H02153

腸内細菌代謝産物に焦点を当てた高食肉摂取による健康増進と病態発症の分岐点の解明
科学研究費助成事業
01 Apr. 2021 - 31 Mar. 2025
清水 英寿, 石塚 敏, 吹谷 智, 田中 愛健, 吉清 恵介, 橋口 亜由未
本研究では、「腸内細菌代謝産物に焦点を当てた高食肉摂取による健康増進と病態発症の分岐点の解明」に向け、腸内で産生されるインドール系化合物の一種、インドール酢酸とその代謝産物であるスカトールを中心に、肝臓および腸管への影響について解析を行っている。
培養肝がん細胞を用いた解析では、スカトールは、その受容体として報告されているAryl hydrocarbon receptor （AhR）の活性化を介するだけでなく、ある核内受容体型転写因子の活性化も介して細胞増殖を導くことが明らかとなった。また我々は、スカトールによって発現増加する長寿遺伝子の1つであるSirt1が、培養肝がん細胞の増殖を導く結果を得た。
培養大腸がん細胞を用いた解析では、スカトールは、Nuclear factor-kappa B（NF-kappaB）の活性化を介してInterleukin-6（IL-6）の発現増加を導いた。またスカトールは、Tumor necrosis factor alpha（TNFalpha）を発現増加させる一方、インドール酢酸に関しては、その発現低下を導くことを我々は報告している。このTNFalphaの発現低下メカニズムとして、TLR4が関与していることが示唆された。さらにインドール酢酸は、AhRの活性化を介して、腸管上皮内腔膜の塩化物-炭酸水素交換体としてNa+/H+交換体とともに腸管での電気陰性NaCl吸収に関与しているsolute-linked carrier 26（SLC26A3）の発現増加を誘導することが明らかとなった。SLC26A3の発現低下は、炎症性腸疾患や大腸癌で観察されることから、インドール酢酸はこれら病態に対する予防および改善に寄与すると考えられる。
日本学術振興会, 基盤研究(B), 島根大学, 21H03356

12水酸化胆汁酸と関連代謝物および腸内細菌叢の解析を基盤とした未病指標の構築　　　　　　　　　　　　　　　
研究助成
2023
公益財団法人 秋山記念生命科学振興財団, Competitive research funding

12α水酸化一次胆汁酸による小腸粘膜バリア脆弱化機構-免疫応答への波及効果-　　　　　　　　　　　　　　　
Jul. 2021 - Jun. 2022
石塚 敏
公益財団法人 三島海雲記念財団, Principal investigator

新規脂肪肝モデルを用いたプレバイオティクスによる肝脂質蓄積抑制作用の検証　　　　　　　　　　　　　　　
学術研究助成金
Apr. 2021 - Apr. 2022
石塚 敏
公益財団法人飯島藤十郎記念食品科学振興財団, Principal investigator

オカラシンバイオティクスによる12α水酸化胆汁酸代謝制御を介する肝脂質蓄積の抑制作用　　　　　　　　　　　　　　　
研究助成
Apr. 2021 - Mar. 2022
石塚 敏
公益財団法人不二タンパク質研究振興財団, 一般研究, Principal investigator

Mechanism of chronic poisoning by exposure to low concentrations of microcystin and verification of mitigation methods in consideration of the intestinal environment
Grants-in-Aid for Scientific Research
29 Jun. 2018 - 31 Mar. 2021
SHIMIZU Hidehisa
In recent years, with the globalization of food, the possibility of ingestion of microcystin, a toxin derived from blue-green algae, through drinking water and freshwater seafood has been pointed out not only in developing countries but also in developed countries. In the present study, we examined the effects of chronic exposure to low concentrations of microcystin on organ function. The results showed that exposure to low concentrations of microcystin induced functional disorders in the liver and colon. Analysis using cultured hepatocytes and intestinal epithelial cells suggested the existence of unconventional pathways of microcystin action. Based on the results of present study, we expect to further elucidate the mechanism of action and search for biomarkers for chronic microcystin poisoning in the future.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Research (Exploratory), Shimane University, 18K19854

Analysis of the disruption and recovery mechanism on gastrointestinal homeostasis by skatole produced by high animal protein intake
Grants-in-Aid for Scientific Research
01 Apr. 2018 - 31 Mar. 2021
SHIMIZU Hidehisa
In recent years, increased intake of red and processed meat in particular has been considered to be one of the factors in the development and progression of liver diseases (fatty liver, liver cancer, etc.) and intestinal diseases (inflammatory bowel diseases, colon cancer, etc.). In the present study, we focused on the tryptophan-derived skatole, an intestinal microbiota metabolite, which is produced in the intestine depending on the amount of protein ingested. While skatole leads to dysfunction of the liver and colon, its precursor, indole-3-acetic acid, was suggested to have the opposite effects. Therefore, the results of the present study indicate that skatole and its precursor indole-3-acetic acid may be involved in the cause of the conflicting effects of increased protein intake on health promotion, and development and progression of diseases that have been reported so far.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Shimane University, 18H03178

脂肪肝の発症及び悪化における12α水酸化胆汁酸の関与　　　　　　　　　　　　　　　
科学研究費補助金
2019 - 2021
石塚 敏
日本学術振興会, 基盤研究(B), Principal investigator, Competitive research funding

新規非アルコール性脂肪肝炎モデルを用いた乳製品・プロバイオティクスによる予防効果の検証　　　　　　　　　　　　　　　
ダノン学術研究助成金 一般研究助成
Apr. 2018 - Mar. 2019
石塚 敏
公益財団法人ダノン健康栄養財団, Principal investigator, Competitive research funding

Influence of intestinal fermentation products caused by excessive animal protein intake on intestinal and various organs.
Grants-in-Aid for Scientific Research
01 Apr. 2015 - 31 Mar. 2018
Shimizu Hidehisa, MIYAZAKI Hitoshi, ISHIZUKA Satoshi
In the present study, we analyzed the influence of skatole, an intestinal bacterial metabolite, on rats, cultured intestinal cells, and cultured hepatoma cells. As results, in rats, it was revealed that skatole induces disturbance of bile acid metabolism, change of gene expression in the liver and ileum, and dysbiosis. In the analysis using cultured intestinal cells, it was demonstrated that skatole leads to cell death via AhR activation. Furthermore, in cultured hepatoma cells, it was suggested that skatole promotes cell proliferation through activation of ERK. Based on the above results of the present study, it was shown that the production of skatole in the intestine induces abnormalities in gastrointestinal function, and may also be involved in the onset and progression of gastrointestinal diseases.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Shimane University, 15H03090

実験動物における「未病」モデルの確立と食品の生理作用評価への応用　　　　　　　　　　　　　　　
一般公募研究
Apr. 2017 - Mar. 2018
石塚 敏
一般財団法人 糧食研究会, Principal investigator, Competitive research funding

胆汁酸負荷により体内で増加する化合物の同定と生活習慣病発症における関与　　　　　　　　　　　　　　　
科学研究費補助金
Apr. 2016 - Mar. 2018
石塚 敏
日本学術振興会, 挑戦的萌芽研究, Principal investigator, Competitive research funding

Novel mechanism analysis of microcystin on organ dysfunction-focusing on bile acid metabolism-
Grants-in-Aid for Scientific Research
01 Apr. 2015 - 31 Mar. 2017
Shimizu Hidehisa, SUGIURA NORIO, MIYAZAKI HITOSHI, ISHIZUKA SATOSHI, FUKITANI SATORU
Eutrophication of lake and reservoir accelerates development of Microcystis which produces a toxin, called microcystin. The present study shows that in a cultured hepatocyte cell line, HepG2 cells, AMPK was time- and dose-dependently activated by microcystin-LR (MC-LR) stimulation. When AMPK was not activated in MC-LR-treated HepG2 cells, cell death was induced. However, when MC-LR activated AMPK, cell death was inhibited. In a cultured intestinal cell line, Caco-2 cells, MC-LR leaded to cell proliferation through p38 and JNK activation although ERK activation was not participate. Furthermore, the expression levels of oncogene was upregulated in MC-LR-treated Caco-2 cells. In rat analysis, food intake, body weight, and weight of liver, kidney, and adipose tissue were not difference between control and MC-LR-administrated rats. We will continue to check for serum parameter (ALT/AST, TG, and TC etc), liver TG and TC, and gut microbiota, etc.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Shimane University, 15K12352

Development of novel food materials for lifestyle disease prevention using edible fungi
Grants-in-Aid for Scientific Research
01 Apr. 2013 - 31 Mar. 2017
KUMURA HARUTO
Monascus species, known as red-mold has the ability to produce diverse functional secondary metabolites such as lovastatin, monascin and ankaflavin which are responsible for health benefits including risk reduction of arteriosclerosis. It would be applicable for development of functional foods, however, some strains of Monascus sp. produce nephrotoxin, citrinin. Therefore, strain and culture condition should be carefully selected. In addition to the selection of test strains, we focused on the materials for culture substrate, which should be solid with convenience for preparation, if necessary, capability to add nutritional supplements, applicability to wide pH range and “ready to eat” property of the culture products. Following the screening of the suitale strain and culture condition, the resulting culture products were fed to experimental animals whether it could exert predicted biological effects.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 25350120

食を介する健康維持の基盤：腸内細菌と脂肪細胞の制御　　　　　　　　　　　　　　　
地域イノベーション戦略支援プログラム さっぽろヘルスイノベーション‘Smart-H’
2012 - 2016
石塚 敏
文部科学省, Principal investigator

Elucidation of mechanisms to avoid chronic inflammation via enteroendocrine system: relationships between immune cells and adipocytes
Grants-in-Aid for Scientific Research
2012 - 2013
ISHIZUKA Satoshi, HIRA Tohru
Diet-induced obesity induces chronic inflammation. At postplandial period, various external antigens other than dietary lipids get into lymph ducts and evoke immune responses. In this study, we evaluated factors in lymph ducts by using lymph-cannulated rats especially at a postplandial period. At several hours after administration of dietary lipids, there was a significant increase of interleukin-6(IL-6) and glucagon-like peptide-1 in the lymph. Gene expression analysis in food-deprived or fed rats indicated that an accumulation of IL-6 producing cells in the intestinal mucosa accompanied by lipid absorption.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, Principal investigator, Competitive research funding, 24658114

Development of an innovative method for reducing colon-cancer-inducing secondary bile acid formation through enhancement of anaerobic respiration of the intestinal bacteria
Grants-in-Aid for Scientific Research
2011 - 2012
YOKOTA Atsushi, ISHIZUKA Satoshi
Some secondary bile acids formed in the large intestine are known as a riskfactor of colon cancer. In this study a novel method that reduces such secondary bile acids formed by thereductive metabolism of gut microbes were investigated. We tested if the enhancement of oxidativemetabolism of the responsible gut microbes by the introduction of anaerobic respiration can reduce thetarget secondary bile acids. Thus, rats were fed sodium fumarate-supplemented diet to enhanceanaerobic respiration of the gut microbes. Although the results suggested a possibility of decreasingrelative abundance of deoxycholic acid, a representative colon-cancer-inducing secondary bile acid, theobserved effects were not practically applicable, because high dose of fumarate was required (5~10% ofthe diet), which resulted in the diarrhea in rats experiments. Therefore, improved method of fumaratefeeding, e.g. encapsulation, might be necessary to achieve this goal.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 23658064

消化管陰窩におけるケモカイン分泌上皮の局在と食餌成分による調節機構
科学研究費補助金
2004 - 2006
石塚 敏
本研究では消化管リンパ球の局在とそれにおよぼす食餌成分の影響についてケモカインの関与を検討した。本研究を通じて明らかになったことは以下の通りである。これまでにもパイエル板を除いたラット小腸粘膜固有層からリンパ球の分離が可能であったが、その分離法を改善することにより回収率を上げる事に成功した。また、その方法を基にしてラット大腸粘膜からのリンパ球分離を可能にした。難消化性糖類は上部消化管で吸収されずに大腸へ流入することで、そこに存在する腸内菌に資化されることを考えれば、大腸粘膜におけるリンパ球分離法の確立は今後の重要な課題を解決するための基盤技術となりうる。食餌成分として甜菜繊維(SBF)を用いた場合、WKAHラット盲腸部位におけるCD8^+上皮間リンパ球(IEL)の頻度を有意に増加させたが、小腸でも同様な現象が観察された。興味深いことに、盲腸に比べ小腸での上昇率の方が極めて高いことが明らかになった。しかし、このSBF摂取による小腸でのCD8^+ IEL増加は、ラットの系統により異なることが示された。DAラットではこのような小腸における増加は全く確認されなかった。このことは遺伝的背景により食餌による消化管粘膜リンパ球局在調節機構が異なることを示している。小腸から機械的に陰窩および絨毛部位を分離し、それぞれRT-PCR解析を行ったところ、この部位間で異なる発現を示すものとしてCCL...
文部科学省, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 16780088

大腸上皮内リンパ球機能の食餌成分による調節機構
科学研究費補助金
2001 - 2002
石塚 敏
昨年度の実験において、難消化性糖類の摂取によりCD8^+大腸上皮間リンパ球(IEL)の大腸粘膜への集積が生理的な範囲で起こることを明らかにした。今年度はそれ以外にCD4^+ IELとNKR-P1A^+大腸上皮間ナチュラルキラー細胞(IENK)の局在を免疫組織化学的に検討した。NKR-P1A^+ IENKは、近年になってラットのIEL画分よりその存在が明らかになった細胞集団で、NK活性を持ち、IFN-gammaとIL-4を分泌する特殊な細胞集団であることが報告されている。興味深いことに、CD8^+ IELは大腸陰窩中において既に分化した上皮細胞間に存在するのに対し、NKR-P1A^+ IENKは逆に増殖上皮細胞の近傍、すなわち陰窩底部に存在することが陰窩内セルポジションごとの解析により明らかになった。また、NKR-P1A^+ IENKの局在はCD8^+ IEL同様に盲腸に多く、以降遠位に向かって減少することも明らかになった。さらに陰窩内におけるNKR-P1A^+ IELの数はそのCD8^+ IELの数と強い正の相関を示した。これらの局在はこれまでの栄養学的な観点からの標準飼料の摂取では見られず、発酵性の難消化性糖類を添加することにより初めて顕在化されることが盲腸内容物のHPLC分析から示唆された。しかし、その集積は病理的なものではないことは組織化学的解析から明らかである。一方、...
文部科学省, 奨励研究(A), 北海道大学, Principal investigator, Competitive research funding, 13760090

Prevention of initiation and promotion of colon cancer by dietary fiber and design of highly functional dietary fiber.
Grants-in-Aid for Scientific Research
1998 - 2000
HARA Hiroshi, ISHIZUKA Satoshi
The aims of this study were to establish a model of colon cancer by ionizing radiation in rats and to compare the effects of dietary fiber feeding on cancer development with those in a chemically-induced colon cancer model. We first succeed to develop precancerous legion, aberrant crypt foci (ACF), in rat colonic mucosa by ionizing radiation. The properties of radiation-induced ACF were low frequency, but high multiplicity compared with the chemically induced ACF Feeding of a highly fermentable dietary fibers, sugarbeet fiber, suppressed ACF in the chemical model, but not the radiation model. In the chemical model, a low fermentable, soluble fiber prevent the development of colonic ACF just feeding fiber before, but not after a challenge of carcinogen. Also, a product of the colonic fermentation, butyric acid, prevented the development of ACF in the rat colon using the chemically-induced cancer model.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B)., GRADUATE SCHOOL OF AGRICULTURE, HOKKAIDO UNIVERSITY, 10460053

新規バイオポリマーの生物機能
科学研究費助成事業
1998 - 1999
青山 頼孝, 石塚 敏
Enterobacter cloacae ECP126が生産した新規バイオポリマーが血清脂質を低下させる能力を有するかどうか検討するため、バイオポリマーをラットの食事に5%添加した。ヒスチジンの添加によって誘導される内因性高コレステロール血症は、新規バイオポリマーの添加によって血清コレステロール、リン脂質のいずれも有意に低下させる作用を有することを見出した。一方、トリアシルグリセロールは低下の傾向を示した。基本食、ヒスチジン食摂取ラットの盲腸内の個々の有機酸量は変わらなかった。基本食にバイオポリマーの添加は盲腸中の酢酸、酪酸レベルを有意に増加させた。一方、ヒスチジン食にバイオポリマーの添加は、基本食にバイオポリマーを添加したときと比較して有機酸の全量は有意に低下した。さらに主要な成分である酢酸は低下の傾向、プロピオン酸は有意に低下したがバイオポリマーを添加しなかったヒスチジン食群と比較して、有機酸の全量、酢酸、プロピオン酸レベルは有意に増加した。これらの事実から、盲腸内のプロピオン酸量は血清コレステロールレベルを調節していると推定された。したがってEnterobacter cloacae ECP126から生成される新規バイオポリマーは粘度が著しく高く、血清コレステロールを低下させる機能を有する成分であり、将来利用されうる高分子化合物であることがあきらかになった。これらの成果は平成11年度日本栄養・食糧学会総会(平成11年5月29日)(東京)において"Nutritional effect of biopolymer produced by microorganism on serum lipids in rats"を口頭発表した。
日本学術振興会, 萌芽的研究, 北海道大学, 10876027

Molecular basis for establishing the environment in digestive tract by food components
Grants-in-Aid for Scientific Research
1998 - 1999
KAWABATA Jun, ISHIZUKA Satoshi, KURIHARA Hideyuki
As the inhibitory principles against rat intestinal maltase, two ellagitannins, eugeniin and casuarictin, were isolated from the methanolic extracts of Clove buds. Structure-activity relationship study of the isolates together with synthetic pentagalloylglucose (PGG), which showed the highest activity , revealed that the inhibitory activity depended on the number of uncoupled galloyl residues in the molecule. These tannins also inhibited maltase expressed on a human carcinoma cell line, Caco-2. On the other hand, the acetone extracts of roselle tea, dried flower buds of Hibiscus sabdariffa, possessed an inhibitory activity against porcine pancreatic α-amylase. The active compound was identified as hibiscus acid.
Inhibitory potencies of the extracts of seafood and algae against Escherichia coli β-glucuronidase were investigated by the colorimetric method in vitro. Among them, the extracts of dried bonito (katsuobushi), the brown alga Hizikia fusiformis, and the red alga Chondria crassicaulis showed the inhibitory activity. The inhibitor obtained from dried bonito was disclosed to be inosinic acid. Among nucleic acid-related compounds, adenylic acid and adenosine triphosphate also inhibited the β-glucuronidase. The inhibitor obtained from H. fusiformis was identified to be a mixture of free fatty acids. Isolation and structural elucidation of the inhibitor of C. crassicaulis is now in progress.
Direct effect of molecules on the formation of aberrant crypt foci (ACF) induced by 1,2-dimethylhydrazine using the intrarectal injection was investigated. Butyric acid or deoxycholic acid (and the derivatives) was used as the test molecule. Intrarectal injection of butyric acid showed a protective effect on the ACF formation at 20 mM. On the other hand, intrarectal injection of deoxycholic acid significantly promoted the size of ACF. These results suggest that the intrarectal injection with experimental animal is useful model to assess the effect of such molecules on the early phase of carcinogenesis.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 10460052

Role of genotoxic stress-induced apoptosis in the protective effect of dietary fiber against tumorigenesis in the large bowel
Grants-in-Aid for Scientific Research
1997 - 1998
KASAI Takanori, ISHIZUKA Satoshi, SONOYAMA Kei
The present study investigated the role of genotoxic stress-induced apoptosis in the protective effect of dietary fiber against tumorigenesis in the large bowel. Dietary beet fiber increased the apoptotic frequency in rat colonic epithelium at 6 h after a subcutaneous injection of carcinogen 1 , 2-dimethylhydrazine (DM11). The finding suggests that increased efficiency of elimination of damaged cells may be associated with the anti-tumorigenic effect of dietary beet fiber. Semi-quantitative RT-PCR demonstrated that increase in the expression of cyclin-dependent kinase inhibitor p21 and apoptosis inducer Bax genes, which are targets of tumor suppressor protein p53, preceded the apoptosis in rat colonic epithelium following DMH injection. In addition, gene expression of betaA-subunit of activin which is a member of TGF43 gene superfamily was also shown to be upregulated, To establish in vitro model for the investigation of these phenomena at cellular and molecular bases, we examined the time-course of changes in cell mortality and gene expression following different doses of UV irradiation in the untransformed cell line IEC-6 which was derived from rat small intestine. Cell survival rates were decreased by UV irradiation in time- and energy-dependent fashion. Agarose gel electrophoresis of cellular DNA demonstrated that the cell death was due to apoptosis. Semi-quantitative RT-PCR revealed the increase in mRNA levels of betaA-subunit of activin in IEC-6 cells following UV irradiation, being similar to that in rat colonic epithelium following DM14 treatment. The next step concerns whether the dietary fiber and its fermentation products short chain fatty acids modulate the cell cycle, apoptosis, and expression of related genes the colonic epithelial cells following genotoxic agents.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), HOKKAIDO UNIVERSITY, 09660123

Physiological and Biochemical Study of Gastrointestinal Functions.
Grants-in-Aid for Scientific Research
1997 - 1998
AOYAMA Yoritaka, ISHIZUKA Satoshi, SONOYAMA Kei, KAWABATA Jun, KASAI Takanori
(1) When rats were fed an orotic acid diet, liver lipid level of rats fed a diet containing casein was significantly higher than that of a diet containing egg protein or soybean protein. The addition of an orotic acid to a diet containing casein caused to decrease liver superoxide dismutase activity, to increase serum ornithine carbamoyl transferase activity, to increase the hepatic concentration of 1,2-diacylglycerol. Liver damage by administration of an orotic acid might be due to the increased concentration of 1,2-diaclglycerol and decreased activity of hepatic superoxide dismutase.
(2) 9-Oxooctadeca 10,12-dienoic acid as acetyl CoA carboxylase inhibitor from red pepper was found. Its activity was 50 fold higher as compared with the normal fatty acids.
(3) Dietary beet fiber had a role to supprees the formation of the aberrant crypt foci induced by 1,2-dimethylhydrazine. Furthermore, it was found that the formation of the aberrant crypt foci induced by nu -ray irradiation was suppressed by the injection of anti-asialo GMI, but not by the addition of beet fiber.
(4) Apoptosis and p53 level in epithelial cell of colon was induced by the administration of 1,2-dimethylhydrazine. Further observation was increased level of activin mRNA in colon. This is the first finding that activin is a factor to stop the cell cycle in apoptosis.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 09306007

食物繊維による大腸上皮細胞の異常増殖抑制作用における免疫系の関与
科学研究費補助金
1997 - 1998
石塚 敏
大腸癌の前癌病変と考えられているaberrant crypt foci(ACF)は発癌物質の投与だけでなく、低線量のガンマ線照射によっても引き起こされることが我々の研究で明らかになった。大腸癌を誘発させるために比較的よく使われる発ガン物質は大腸管腔内側から作用するものが多い。一方、ガンマ線の場合は直接大腸上皮に作用し、かつ照射時間も数分であるため、比較的腸管腔内容物の影響を受けにくいと考えられる。我々はこのガンマ線誘発ACFが抗アシアロGM1投与により影響されるか、また大腸癌誘発剤である1,2-dimethylhydrazine(DMH)誘発ACFを強力に抑制した甜菜食物繊維(SBF)の摂取がガンマ線誘発ACFに対しても同様な効果を持つのかを検討した。その結果、DMH誘発ACFの場合と同様に抗アシアロGM1の投与はガンマ線誘発ACFの発生頻度を有意に増加させることが明らかになった。SBFの摂取自体はガンマ線誘発ACFに対して影響しないが、抗アシアロGM1投与下ではSBF摂取はACFの増加を有意に抑えた。また、免疫組織化学的研究から、抗アシアロGM1投与により大腸粘膜上皮内におけるCD8陽性細胞の数が有意に減少することが明らかになった。これは抗アシアロGM1投与は大腸上皮間リンパ球に影響を与えることを示唆している。これらの研究から粘膜上皮近傍のCD8陽性細胞数が減少した条件で、...
文部科学省, 奨励研究(A), 北海道大学, Principal investigator, Competitive research funding, 09760120

二次胆汁酸の生成抑制剤　　　　　　　　　　　　　　　
Patent right
特許公開2004-244365

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石狩管内教育研究会 栄養教諭部会
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北海道新聞社編集局・北海道大学農学部
あぐり大学
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