Ryuhei Kanamoto

Evidence for the existence of a soybean resistant protein that captures bile acid and stimulates its fecal excretion.

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.

Elimination of Na+-dependent bile acid transporter from small intestine by ileum resection increase colonic tumorigenesis in the rat fed deoxycholic acid

Abstract Ileal Na+-dependent bile acid transporter (ISBT) constituting a gateway to enterohepatic circulation of bile acids occurs exclusively at the distal site of the small intestine. In the present study, we examined colonic tumorigenesis promoted by deoxycholic acid in relation to the expression of the ISBT. For this purpose, the small intestine of a Fischer-344 rat was resected a length of 20 cm above the ileo-cecal valve (ileal resection) or below the duodenum (jejunal resection). Then, rats were treated with an intraperitoneal injection of azoxymethane (15 mg/kg body wt.) once a week for 3 weeks and fed a 20% casein diet supplemented with 0.2 % deoxycholate for 39 weeks. Northern blot analysis demonstrated that the ISBT mRNA was hardly detectable in ileum-resected rats. The excretion of fecal bile acids was 1.5-fold higher in the ileum-resected group than in the jejunum-resected group (P

Response of the Induction of Rat Liver Serine Dehydratase to Changes in the Dietary Protein Requirement

Growing and mature rats were examined for the effect of a change in dietary protein requirements on the induction of liver serine dehydratase (SDH). The rats were fed on diets varying in casein content, and the weight change and nitrogen balance was determined. SDH activity and its gene expression were induced in both growing and mature rats when their protein intake exceeded their nutritional requirements.

Deoxycholic Acid Can Induce Apoptosis in the Human Colon Cancer Cell Line HCT116 in the Absence of Bax

In the human colon cancer cells HCT116, deoxycholic acid (DCA) induces apoptosis via the mitochondrial pathway by triggering the release of mitochondrial factors such as cytochrome c. To elucidate if Bax, a proapoptotic member of the Bcl-2 family known to trigger cytochrome c release in response to various types of apoptotic stimuli, is involved in DCA-induced apoptosis in HCT116 cells, we analyzed DCA-induced apoptosis in Bax-knockout (Bax–/–) HCT116 cells. Cytochrome c release and caspase-9 activation were detectable after 5 min in both Bax–/– and Bax+/– HCT116 cells. Caspase-3 and caspase-8 activation was observed after 15 and 30 min, respectively. Bax–/– cells were protected from apoptosis by treating them with ursodeoxycholic acid for 12 h prior to DCA treatment. These results are consistent with our previous observations that were obtained by using wild-type HCT116 cells and suggest that Bax is not indispensable for DCA-induced apoptosis in HCT116 cells.

Biphasic Regulation of Cell Death and Survival by Hydrophobic Bile Acids in HCT116 Cells

A secondary bile acid, namely, deoxycholic acid (DCA), has been known to promote colon tumors; on the other hand, it also induces apoptosis in several human colon cancer cell lines. A hydrophobic primary bile acid, namely, chenodeoxycholic acid (CDCA), exhibits a similar property of apoptosis induction; DCA and CDCA also trigger some specific intracellular signal pathways in the human colon cancer cell line HCT116. In this article, we report that hydrophobic bile acids induce different cellular responses depending on their concentration, that is, a sublethal concentration of hydrophobic bile acids can suppress the apoptosis induced by a higher concentration of DCA. Pretreatment with DCA or CDCA at a concentration of ≤ 200 μ M for 8 h suppressed the apoptosis induced by 500 μ M DCA in HCT116 cells. Under this condition, the association of caspase-9 and Apaf-1 and subsequent activation of caspase-9 were inhibited, but the release of cytochrome c from the mitochondria was not. At 200 μ M, DCA and CDCA induced the phosphorylation of Akt and ERK1/2, although these phosphorylations do not appear to be indispensable for the cytoprotection. It is interpreted that prolonged exposure to sublethal concentrations of hydrophobic bile acids induces resistance to apoptosis, leading to promotion of colorectal tumorigenesis.

Characterization of cloned mouse Na+/taurocholate cotransporting polypeptide by transient expression in COS-7 cells.

The mouse Na+/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [3H]taurocholic acid with Km and Vmax values of 18 μM and 102 pmol/mg protein/min, respectively. This Km value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [3H]taurocholic acid transport.

Ursodeoxycholic Acid Protects Colon Cancer HCT116 Cells From Deoxycholic Acid-Induced Apoptosis by Inhibiting Apoptosome Formation

We previously demonstrated that ursodeoxycholic acid (UDC) requires prolonged (≥5 h) preincubation to exhibit effective protection of colon cancer HCT116 cells from deoxycholic acid (DC)-induced apoptosis. Although UDC diminished DC-mediated caspase-9 activation, cytochrome c release from the mitochondria was not inhibited, indicating that UDC acts on the steps of caspase-9 activation. In the present study, therefore, we investigated the effects of UDC on the factors involved in caspase-9 activation. We found that UDC had no significant effect on the expression of antiapoptotic XIAP. Furthermore, UDC did not affect the expression or release of proapoptotic Smac/DIABLO, or the association of XIAP and Smac/DIABLO. In contrast, association of Apaf-1 and caspase-9 stimulated by 500 μM DC was inhibited by UDC pretreatment. Although UDC caused remarkable activation of Akt/PKB, phosphatidylinositol-3-kinase (PI3K) inhibitor did not significantly reduce UDC-mediated cytoprotection. Furthermore, phosphorylation of threonine residues on caspase-9 after UDC pretreatment could not be detected. UDC-mediated cytoprotection was independent of the MAPK pathway, and cyclic AMP (cAMP) analogue did not inhibit DC-induced apoptosis. Our results indicate that UDC protects colon cancer cells from apoptosis induced by hydrophobic bile acids, by inhibiting apoptosome formation independently of the survival signals mediated by the PI3K, MAPK, or cAMP pathways.

Effects of Cys mutation on taurocholic acid transport by mouse ileal and hepatic sodium-dependent bile acid transporters.

All cysteines of mouse ileal and hepatic sodium-dependent bile acid transporters (Isbt and Ntcp, respectively) were individually replaced by alanine. Replacement of Cys106 in Isbt and Cys96 in Ntcp, which are located closely in alignment, decreased taurocholate uptake. Although Cys51 in Isbt is conserved in Ntcp, the replacement spoiled Isbt only. Both similarity and difference in the arrangement of functional sites are suggested.

Soybean Resistant Protein Elevates Fecal Excretion of Cholesterol and Bile Acids and Decreases Hepatic Cholesterol Content in Comparison with Soybean Protein Isolate

The effect of soybean resistant protein (RP) on serum and hepatic cholesterol levels and fecal excretion of steroids was examined. RP decreased cholesterol in the liver, probably due to the stimulated excretion of cholesterol and its metabolites, bile acids. The serum cholesterol level was not different as between RP and other soy-derived proteins.

Inverse Correlation between the Nitrogen Balance and Induction of Rat Liver Serine Dehydratase (SDH) by Dietary Protein

Rats of different ages (3 to 15-wk-old) were fed on a 25% casein diet for one week, and the nitrogen balance and liver serine dehydratase (SDH, EC 4.2.1.13) activity were then determined. The value for nitrogen balance decreased with the age of the rats, while the liver SDH activity increased. A statistical analysis showed clear inverse correlation between the two factors (R2 = 0.7372, p < 0.01). This result suggests that SDH was induced by response to the amount of surplus amino acids from dietary protein taken beyond the body’s requirement. The increase in SDH activity was accompanied by an increase in the level of SDH mRNA. Since the half-life of this mRNA did not change significantly, the induction was mainly controlled at the level of transcription. In addition, the induction seems not to be related to gluconeogenesis, since the mRNA levels of tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), other gluconeogenic enzymes, were not changed under these experimental conditions.