Hirohito Watanabe

Aquaporin-9 is expressed in a mucus-secreting goblet cell subset in the small intestine

We analyzed the expression of aquaporins (AQPs) in the small intestine to elucidate their functions, and found that AQP9, which had not previously been detected there, is present in duodenum, jejunum, and ileum. AQP9 is expressed in colon as well, but not in stomach. Also, its expression in these intestinal sections is limited to the basolateral membranes of a goblet cell subset. Our finding that AQP9 is present specifically in goblet cells as mucus‐secreting cells suggests its involvement in the synthesis and/or secretion of a certain kind of mucus which may protect the intestinal surface and smooth the flow of intestinal contents.

Identification and Determination of α-Dicarbonyl Compounds Formed in the Degradation of Sugars

The α-dicarbonyl compounds formed in the degradation of glucose and fructose were analyzed by HPLC using 2,3-diaminonaphthalene as derivatizing reagent, and identified as glucosone (GLUCO), 3-deoxyglucosone (3DG), 3-deoxyxylosone (3DX), tetrosone (TSO), triosone (TRIO), 3-deoxytetrosone (3DT), glyoxal (GO), and methylglyoxal (MGO). The results suggest that α-dicarbonyl compounds were formed from glucose via non-oxidative 3-deoxyglucosone formation and oxidative glucosone formation in glucose degradation. In addition, TRIO, GO, and MGO were also formed from glyceraldehyde as intermediate. The α-dicarbonyl compounds might be formed from glucose via these pathways in diabetes.

Cytotoxicity and Oxidative Stress Induced by the Glyceraldehyde-related Maillard Reaction Products for HL-60 Cells

We demonstrated the cytotoxicity of glyceraldehyde-related Maillard reaction products for HL-60 cells. Glyceraldehyde-modified bovine serum albumin and glyceraldehyde-modified casein inhibited the proliferation of HL-60 cells. The reaction products formed from glyceraldehyde and Nα-acetyllysine had also a cytotoxic effect on HL-60 cells. The cytotoxic effect was prevented by N-acetylcysteine or pyrrolidinedithiocarbamate as the antioxidants. In addition, the reaction products depressed the intracellular glutathione level, and induced the reactive oxygen species (ROS) production. These results suggested that the glyceraldehyde-related advanced glycation end products (AGEs) induced the cytotoxicity and the oxidative stress. We previously reported that the glyceraldehyde-related AGE was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named GLAP (glyceraldehyde-derived pyridinium compound), formed from glyceraldehyde and Nα-acetyllysine (Biosci. Biotechnol. Biochem., 67, 930–932 (2003)). In this study, GLAP inhibited the proliferation of HL-60 cells, and the inhibitory effect was prevented by the antioxidants. Furthermore, GLAP depressed the intracellular glutathione level, and induced the ROS production. This work indicated the possibility that the cytotoxicity and the oxidative stress in the progression of diabetic complications and chronic renal disease might be induced by GLAP.

Detection and determination of glyceraldehyde-derived pyridinium-type advanced glycation end product in streptozotocin-induced diabetic rats.

GLAP, glyceraldehyde-derived pyridinium-type advanced glycation end product (AGE), formed by glyceraldehyde-related glycation, was identified in the plasma protein and the tail tendon collagen of streptozotocin (STZ)-induced diabetic rats. It was detected in the plasma protein and the collagen in diabetic rats by LC-MS and LC-MS/MS analysis, but was not detected in normal rats. In addition, GLAP was formed from glyceraldehyde-3-phosphate (GA3P) with lysine as well as glyceraldehyde (GLA) with lysine in vitro. Accordingly, it is suggested that an increase in the GLAP level reflects an increase in the GLA level and the GA3P level. GLAP might be a biomarker for reduced activity of the glyceraldehyde-related enzymes in the metabolic diseases such as diabetic complications.

Non-Involvement of the Human Monocarboxylic Acid Transporter 1 (MCT1) in the Transport of Phenolic Acid

Phenolic acids such asp-coumaric acid and microbial metabolites of poorly absorbed polyphenols are absorbed by the monocarboxylic acid transporter (MCT)-mediated transport system which is identical to the fluorescein/H+ cotransport system. We focus here on the physiological impact of MCT-mediated absorption and distribution. We examined whether MCT1, the best-characterized isoform found in almost all tissues, is involved in this MCT-mediated transport system. The induction of MCT1 expression in Caco-2 cells by a treatment with sodium butyrate (NaBut) did not increase the fluorescein permeability. Moreover, the transfection of Caco-2 cells with an expression vector encoding MCT1 caused no increase in either the permeability or uptake of fluorescein. Furthermore, in the MCT1-expressing oocytes, no increase ofp-coumaric acid uptake was apparent, whereas the uptake of salicylic acid, a substrate of MCT1, nearly doubled. Our data therefore establish that MCT1 was not involved in the MCT-mediated transport of phenolic acids.

Formation Mechanisms of Melanoidins and Fluorescent Pyridinium Compounds as Advanced Glycation End Products

The formation mechanisms of melanoidins as advanced glycation end products (AGEs) have not been resolved. Blue and red pigments generated in the D‐xylose–glycine reaction system are postulated to be intermediate oligomers in the generation of melanoidins. A novel blue pigment, designated blue‐M5, was identified as a similar structure to blue‐M1 except for the side chain of two dihydroxypropyl groups. Blue pigments were also generated in the D‐glucose–glycine and D‐xylose–β‐alanine reaction systems as well as in the D‐xylose–glycine reaction system. Blue pigments by the Maillard reaction might be formed by the decarboxylation of two molecules of pyrrolopyrrole‐2‐carbaldehydes (PPA). PPA, composed of a side chain of a dihydroxypropyl group, was identified as a precursor of blue pigments. In fact, blue‐M5 was generated by the incubation of PPA alone. Blue pigments, which involved pyrrolopyrrole structures, were readily changed to brown polymers. Glyceraldehyde‐derived pyridinium (GLAP) compound, a glyceraldehyde‐derived fluorescent AGE, and lysyl‐pyrropyridine, a 3‐deoxyglucosone‐derived fluorescent AGE, were detected at higher levels in the plasma proteins and the tail tendon collagen of streptozotocin‐induced diabetic rats compared to normal rats. GLAP and lysyl‐pyrropyridine, therefore, might be related to the progression of diabetic complications.

Identification of a novel blue pigment as a melanoidin intermediate in the D-xylose-glycine reaction system

Some blue pigments were formed in the D-xylose (1 M)-glycine (0.1 M) reaction system. A novel blue pigment, designated as Blue-M2 (blue Maillard intermediate-2), was identified as 5-[1,4-dicarboxymethyl-5-(2,3-dihydroxypropyl)-1,4-dihydropyrrolo[3,2-b]pyrrole-2-ylmethylene]-1,4-dicarboxymethyl-2-{5-[N-carboxymethyl(2,3,4-trihydroxytetrahydrofuran-2-yl)methylamino]-2-hydroxymethyl-4-(1,2,3-trihydroxypropyl)tetrahydrofuran-3-yl}-4,5-dihydropyrrolo-[3,2-b]pyrrole-1-ium. Blue-M2 is presumed to have been generated by the reaction between Blue-M1, which was identified as the major blue pigment in a previous paper (Hayase et al., Biosci. Biotechnol. Biochem., 63, 1512–1514 (1999)), and di-D-xyluloseglycine. Blue pigments are important Maillard reaction intermediates through the formation of melanoidins.

Chemistry and biological effects of melanoidins and glyceraldehyde-derived pyridinium as advanced glycation end products.

Abstract: Blue pigments (blue‐M1 and blue‐M2) and red pigments (red‐M1 and red‐M2) were generated in a xylose‐glycine reaction system. Blue‐M2 was identified as an addition compound of di‐xylulose‐glycine to blue‐M1 that involved two pyrrolopyrrole structures. We identified red pigments as isomers of addition compounds of xylulose‐glycine to the condensed compound between pyrrolopyrrole‐2‐carbaldehyde and pyrrole‐2‐carbaldehyde. These pigments have polymerizing activity, suggesting that they are important Maillard reaction intermediates through the formation of melanoidins. Melanoidins induced IFN‐γ and IL‐12 expression in spleen cells exposed to allergen and in macrophages, respectively. These findings suggest that melanoidins have a suppressive effect on allergic reaction as a novel physiological effect. On the other hand, we identified a glyceraldehyde‐derived advanced glycation end product (AGE) formed from glyceraldehyde and N‐acetylarginine as well as glyceraldehyde‐derived pyridinium (GLAP) in physiological conditions. The AGE was identified as 5‐methylimidazoline‐4‐one (MG‐H1), which has been reported to be formed from arginine and methylglyoxal. GLAP, which induced reactive oxygen species (ROS) production in HL‐60 cells, is supposed to be a toxic AGE, while MG‐H1 is a nontoxic AGE.

Identification of red pigments formed in a D-xylose-glycine reaction system.

Blue, red, and yellow pigments were formed in the D-xylose (1 M)-glycine (0.1 M) reaction system. Novel red pigments were isolated and purified from the reaction solution, designated Red-M1 (red Maillard intermediate-1) and Red-M2 (red Maillard intermediate-2). Red-M1 was identified as 1,4,6,9-tetracarboxymethyl-5-(1,2,3,4-tetrahydroxybutyl)-8-hydroxymethyl-3-(2,3-dihydroxypropyl)-5,6-dihydro-pyrrolo[2′,3′:4,5]pyrrolo[2,3-e]pyrrolo[3,2-b]azepine-9-ium. NMR and CD data indicated that Red-M2 was a diastereomer of Red-M1. They are assumed to be important Maillard reaction intermediates through the formation of melanoidins as well as blue pigments.

Protective Effect of Blue-M1 against Oxidative Stress on COS-1 Cells

Blue-M1 is a blue pigment formed from xylose and glycine in the Maillard reaction. Previous work revealed that Blue-M1 scavenged hydroxyl radicals, and prevented the autoxidation of linoleic acid in vitro. We investigated the protective effect of Blue-M1 for 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH)-induced toxicity in COS-1 cells. COS-1 cells were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37°C for 24 h. Blue-M1 decreased the AAPH-induced toxicity in COS-1 cells, and this effect was dose-dependent. Furthermore, COS-1 cells were treated with diphenyl-1-pyrenylphosphine (DPPP), as a reagent for the detection of lipid peroxide, and then were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37°C for 6 h. Blue-M1 prevented the AAPH-induced peroxidation of cell membrane on COS-1 cells, and this effect was also dose-dependent. These results suggest that Blue-M1 prevents the oxidative cell injury. Therefore, Blue-M1 will be an antioxidant, which protect against the oxidative stress in living systems.