Hitoshi Mineo

Short-chain fatty acids enhance diffusional Ca transport in the epithelium of the rat cecum and colon

We examined the effect of short-chain fatty acids (SCFAs) on Ca absorption from the large intestine in rats in vitro. An Ussing-type chamber technique was used to determine the net transport of Ca from the luminal side to the basolateral side of isolated epithelium in cecum and colon preparations. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. Both solutions were warmed at 37 degrees C and bubbled with 95% O2 and 5% CO2. During and after the incubation period (30 min or 60 min), the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was evaluated. The addition of 80 mM acetic acid, 40 mM propionic acid and 10 mM butyric acid to the mucosal medium increased net Ca absorption (about 300%) in the cecum and colon. An individual application of acetic, propionic or butyric acid (0.01 to 100 mM) to the mucosal medium also increased net Ca absorption at doses of 10 mM and /or 100 mM in the cecum and colon. An increase in solute concentration in the mucosal medium by addition of glycerol or PGE400, or a decrease in pH (7.0-3.0) by addition of HCl did not affect transepithelial Ca transport. We concluded that SCFAs affect the epithelial tissue and promote Ca absorption from the large intestine in vitro. The enhancement of Ca transport induced by SCFAs might be involved in the paracellular transport mechanism.

Indigestible disaccharides open tight junctions and enhance net calcium, magnesium, and zinc absorption in isolated rat small and large intestinal epithelium

The effects of three indigestible disaccharides on net calcium (Ca), magnesium (Mg), and zinc (Zn) transport in isolated rat jejunal, ileal, cecal, and colonic epithelium were determined. Permeability of fluorescein isothiocynate–dextran-4 (FD4) and transepithelial electrical resistance (TEER), which vary according to tight junction (TJ) activity in the intestinal mucosa, were also determined. The addition of 1–100 mM melibiose, difructose anhydride (DFA) III, or DFA IV to the mucosal medium increased the net absorption of the three minerals and FD4 permeability, while decreasing TEER dose dependently in the four intestinal portions. Positive linear relations were found between the net transport of the three minerals and FD4 passage in all portions of the intestine, whereas negative linear relations were observed between net absorption of the three minerals and TEER. We concluded that the three indigestible saccharides directly affect the epithelial tissue and open TJs, thereby promoting Ca, Mg, and Zn absorption in the small and large intestine in vitro.

Newly developed primary culture of rat visceral adipocytes and their in vitro characteristics

We have recently developed a primary culture system for visceral adipocytes (VAs) using stomal‐vascular cells (SVCs) isolated from the mesenteric fat tissue of male Sprague—Dawley rats of 3–5 weeks of age. Modified Dulbeccos modified Eagle medium (DMEM)/F12 containing 17 μM pantothenic acid, 33 μM biotin, 100 μM ascorbic acid, 1 μM octanoic acid, 50 nM triiodothyronine, 10 μg/ml insulin, 10% newborn calf serum (NCS), 100 units/ml penicillin and 100 μg/ml streptomycin was used as a basal culture medium, which did not contain any synthetic compounds usually used to promote adipogenesis, such as indomethacin, dexamethasone, or peroxisome proliferator‐activated receptor (PPAR)‐γ agonists. The SVCs differentiated and proliferated efficiently, and formed a confluent monolayer in 3 days. The VAs accumulated lipids droplets in their cytoplasm at ∼7 days. The differentiation rate from applied SVCs to mature adipocytes was >80% per culture. Adiponectin concentration in the medium increased from Day 5 to Day 7. Application of lipid emulsion stimulated maturation of the SVCs into VAs, as well as subsequent lipid accumulation. Norepinephrine (2 × 10−5 mM) reduced the size of lipid particles and decreased triglyceride (TG) content in the matured adipocytes at 30 min. These results indicate that the new culture system is sufficient to maintain the physiological activity of visceral adipose tissue similar to that in vivo, making it an appropriate and useful tool for basic and applied research on obesity.

Absorptive Activity of Calcium in the Isolated Cecal Epithelium Adaptively Increased by 2 Week's Feeding of Difructose Anhydride III in Rats

We compared net Ca absorption and Lucifer Yellow (LY), a paracellular passage dye, permeability in the epithelium isolated from the rat small intestine, cecum, and colon after feeding with control and difructose anhydride (DFA) III diets for 14 days using the Ussing chamber system. Feeding of DFA III increased net Ca transport and LY passage in the cecal but not in small intestinal or colonic epithelium. Ability of paracellular Ca passage via Tight-junction (TJ) in the cecum was changed adaptively by feeding of DFA III. Changes in microbial fermentation may affect the functional changes of Ca transport in cecal epithelium itself.

Sugar Alcohols Enhance Calcium Transport from Rat Small and Large Intestine Epithelium In Vitro

We compared the effect of a variety of sugar alcohols on calcium absorption from the rat small and large intestine in vitro. An Ussing chamber technique was used to determine the net transport of Ca across the epithelium isolated from the jejunum, ileum, cecum, and colon of rats. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. The Ca concentration in the serosal medium was determined after incubation for 30 min and the net Ca absorption was evaluated. The addition of 0.1–200 mM erythritol, xylitol, sorbitol, maltitol, palatinit, or lactitol to the mucosal medium affected net Ca absorption in the intestinal preparations. Differences in Ca transport were observed between portions of the intestine, but not between sugar alcohols tested. We concluded that sugar alcohols directly affect the epithelial tissue and promote Ca absorption from the small and large intestine in vitro.

Thiazolidinediones exhibit different effects on preadipocytes isolated from rat mesenteric fat tissue and cell line 3T3-L1 cells derived from mice

The effects of PPAR‐γ agonists, thiazolidinediones (TZDs), on preadipocytes isolated from rat mesenteric adipose tissue and murine cell line 3T3‐L1 were compared using an in vitro cell culture system. After each cell formed a confluent monolayer under appropriate medial conditions, pioglitazone or troglitazone was applied at 10 μM to each medium for cell maturation. We observed morphological changes in each cell, especially the accumulation of lipid droplets in the cytoplasm, during the culture periods. At the end of culture, DNA content, triglyceride (TG) content and glycerol‐3‐phosphate dehydrogenase (GPDH) activity were determined. Adiponectin concentrations in each culture medium were also measured during appropriate experimental periods. Application of TZDs increased the DNA content, TG accumulation and GPDH activity in the 3T3‐L1 cells but not in the mesenteric adipocytes. Although TG accumulation was unchanged, the number of lipid particles was decreased and the size of lipid particles in the mesenteric adipocytes was increased by TZD application. Although the TZDs increased adiponectin release from the 3T3‐L1 cells, adiponectin release from mesenteric adipocytes was suppressed (P < 0.05). Thus, the effects of TZDs differed between the primary culture of mesenteric adipose cells and the line cell culture of 3T3‐L1 cells. The source of adipocytes is an important factor in determining the action of TZDs in vitro, and particular attention should be paid when evaluating the effect of PPAR‐γ agonists on adipose tissues.

A Soluble Flavonoid-glycoside, αG-Rutin, Is Absorbed as Glycosides in the Isolated Gastric and Intestinal Mucosa

We investigated the absorption and metabolism of the highly soluble quercetin glycoside αG-rutin, a glucose adduct of insoluble rutin, using the isolated mucosa of the rat stomach and intestines equipped with the Ussing chamber. αG-rutin and rutin appeared in the serosal sides of the gastric body and all the intestinal mucosa after the addition of αG-rutin (1 mM) to the mucosal fluid. The degree of αG-rutin appearance was much lower in the gastric fundus than in the other parts. Quercetin was not found in the mucosal fluid of any mucosal specimen. The concentrations (μM) of αG-rutin and rutin in the serosal fluid as a result of transport from the mucosal side increased time-dependently and linearly with mucosal αG-rutin concentration (1, 10 or 100 mM). The highest transport was shown in the ileal mucosa. These results indicate that αG-rutin is partly hydrolyzed to rutin through the intestine and absorbed as such.

Dicarboxylic acids with limited numbers of hydrocarbons stabilize cell membrane and increase osmotic resistance in rat erythrocytes.

We examined the effect of dicarboxylic acids having 0 to 6 hydrocarbons and their corresponding monocarboxylic or tricarboxylic acids in changing the osmotic fragility (OF) in rat red blood cells (RBCs). Malonic, succinic, glutaric and adipic acids, which are dicarboxylic acids with 1, 2, 3 and 4 straight hydrocarbons located between two carboxylic groups, decreased the OF in a concentration-dependent manner. Other long-chain dicarboxylic acids did not change the OF in rat RBCs. The benzoic acid derivatives, isophthalic and terephthalic acids, but not phthalic acid, decreased the OF in a concentration-dependent manner. Benzene-1,2,3-tricarboxylic acid, but not benzene-1,3,5-tricarboxylic acid, also decreased the OF in rat RBCs. On the other hand, monocarboxylic acids possessing 2 to 7 straight hydrocarbons and benzoic acid increased the OF in rat RBCs. In short-chain dicarboxylic acids, a limited number of hydrocarbons between the two carboxylic groups are thought to form a V- or U-shaped structure and interact with phospholipids in the RBC membrane. In benzene dicarboxylic and tricarboxylic acids, a part of benzene nucleus between the two carboxylic groups is thought to enter the plasma membrane and act on acyl-chain in phospholipids in the RBC membrane. For dicarboxylic and tricarboxylic acids, limited numbers of hydrocarbons in molecules are speculated to enter the RBC membrane with the hydrophilic carboxylic groups remaining outside, stabilizing the structure of the cell membrane and resulting in an increase in osmotic resistance in rat RBCs.

Ingestion of potato starch containing esterified phosphorus increases alkaline phosphatase activity in the small intestine in rats

Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters and plays an important role in phosphorus (P) metabolism. Several nutrients in food have been reported to affect intestinal ALP activity in animal models. Previous reports indicated that high levels of P or phosphate in diets decreased intestinal ALP activity in rats. Because potato starch contains considerable amounts of esterified P, unlike other starch-derived plants, we hypothesized that the feeding of potato starch would decrease ALP activity in the intestinal tract. Male Sprague-Dawley rats (7 weeks old) were fed 3 different types of diet containing 60% corn starch or 1 of 2 types of potato starch with different esterified P content for 1 or 5 weeks. Body weight and food intake of each rat were measured every day throughout the experimental periods. At the end of the feeding periods, the small intestine was removed to determine ALP activity in the mucosal tissues. Significant differences were observed in ALP activity in the small intestine between the 2 feeding periods, among the 4 segments of the small intestine, and among the 3 diet groups. Significant positive linear correlations between the amount of P derived from the starch and mucosal ALP activity were obtained in the jejunum and jejunoileum in rats after feeding for 5 weeks. We concluded, contrary to our hypotheses, that the ingestion of potato starch adaptively increases ALP activity in the upper part of the small intestine of growing rats in an esterified P content-dependent manner.

Effect of the chemical specificity of benzoic acid and its analogs on osmotic fragility in erythrocytes of Sprague-Dawley rats in vitro

We examined the chemical specificity of benzoic (benzene-carboxylic) acid and its derivatives in increasing osmotic fragility (OF) in rat red blood cells (RBCs) in vitro. Benzoic acid increased the OF in the rat RBCs in a dose-dependent manner. Replacement of the carboxylic group with a phosphoric group also increased the OF in RBCs, whereas substitution of the carboxylic group by a sulfonic, amide or hydroxy group did not affect the OF. Replacement of the benzene nucleus with a cyclohexane ring or a straight hydrocarbon chain with six carbons resulted in a greater increase in OF than that induced by benzoic acid. Introduction of a methyl group, chloride or bromide at the m- and p-positions of the benzene ring considerably enhanced the increase in OF induced by benzoic acid. Substitution of the amino and hydroxy group at the m- and p- positions abolished the increase in OF induced by benzoic acid. The introduction of these elements at the o-position showed an almost equal increase in OF as that observed for benzoic acid. A molecule of benzoic acid is composed of both hydrophilic (carboxylic group) and hydrophobic (benzene ring) components. Replacement of the hydrophilic component changed the balance formed between hydrophobic and hydrophilic components in the moiety, resulting alterations to its interaction with the RBC membrane. The size, form and elements introduced into the benzene ring also affected its affinity to the cell membrane, and changed the osmotic resistance in rat RBCs.