Kimikazu Iwami

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.

Deamidation-induced fragmentation of maize zein, and its linked reduction in fatty acid-binding capacity as well as antioxidative effect

Abstract Zein lost its antioxidative effect in proportion to the degree of deamidation. Changes in molecular weight distribution of the protein fraction by deamidation were examined by means of gel filtration with Sephacry S-200 and SDS-electrophoresis in 12% polyacrylamide gel. The deamidation reaction (mild acid-hydrolysis by 0.05N HCl in 70% ethanol) was accompanied by fragmentation of zein subunits. Concomitantly, the surface hydrophobicity as well as fatty acid-binding capacity decreased with progressed deamidation. There was a close correlation ( r = 0.98) between the antioxidative effect and the fatty acid-binding capacity, but not the surface hydrophobicity. A large part of the antioxidative effect of zein is directly or indirectly attributable to its capacity of burying unsaturated lipid in the inter- or intra-molecular hydrophobic spaces in which the amide groups are intimately involved.

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.

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.

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.

Synthesis of pyridoxine-β-glucoside by rice bran β-glucosidease and its in situ absorption in rat small intestine

Abstract A major component of β-glucosidase multi-enzymes was highly purified from rice bran, and by its use, PIN-β-G was synthesized from p-nitrophenyl-β-glucoside and pyridoxine. The synthetic product contained both 4′- and 5′-isomers, which were successfully separated by high-voltage paper electrophoresis. The 4′-isomer was used as a convenient subsrate for intestinal absorption of PIN-β-G, because it gave a positive reaction with 2,6-dibromoquinone chlorimide irrespective of the presence or absence of borate. The absorption experiments with in situ isolated rat jejunal loops revealed that the PIN-β-G level remaining in the loop did not significantly change within an hour and that δ-gluconolactone, a potent inhibitor of β-glucosidase, did neither affect the luminal disappearance of PIN-β-G. It thus can be assumed that PIN-β-G is absorbed across the intestinal wall by a mechanism of simple diffusion, but not of “hydrolase-mediated transport”.

Mutational Analysis of Uncharged Polar Residues and Proline in the Distal One-Third (Thr130–Pro142) of the Highly Conserved Region of Mouse Slc10a2

Solute carrier family member 2 (SLC10A2) reabsorbs bile acids at the distal terminus of the ileum in an Na+-dependent manner. Alignment of deduced amino acid sequences of SLC10 family members and homologous genes in various species revealed a highly conserved region that corresponds to Gly104–Pro142 of SLC10A2. To elucidate the functional importance of this region, uncharged polar residues and Pro in the distal one-third of this region in mouse Slc10a2 (mSlc10a2) were submitted to mutational analysis, and taurocholic acid uptake and cell surface localization were evaluated. In addition to mutations that abolished almost all of the transport activity with and without cellular localization failure (P142V and T130A respectively), a mutation that perhaps affected affinity for taurocholic acid was identified (T134A). These results suggest that the highly conserved region contains residues involved in the substrate interaction, function, and cellular localization of mSlc10a2.