Toshio Taira

Effect of Prolyl-hydroxyproline (Pro-Hyp), a Food-Derived Collagen Peptide in Human Blood, on Growth of Fibroblasts from Mouse Skin

We examined the effect of prolyl-hydroxyproline (Pro-Hyp), which occurs in human peripheral blood after ingestion of collagen peptide, on the migration and growth of mouse skin fibroblasts. Mouse skin discs were cultured on a 24-well plastic plate in a fetal bovine serum (FBS)-free medium. Addition of Pro-Hyp (200 nmol/mL) significantly increased the number of fibroblasts migrating from the skin to the plate after incubation for 72 h. This effect of Pro-Hyp was abolished by the addition of mitomycin C. The fibroblasts that had migrated from the mouse skin were collected and cultured on collagen gel. The growth of fibroblasts on the collagen gel was suppressed even in the presence of FBS, while rapid fibroblast growth was observed on the plastic plate. Addition of Pro-Hyp (0-1000 nmol/mL) to the medium containing 10% FBS enhanced the growth of fibroblasts on the collagen gel in a dose-dependent manner. These results suggest that Pro-Hyp might stimulate the growth of fibroblasts in the skin and consequently increase the number of fibroblasts migrating from the skin.

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.

Bacterial cell wall components regulate adipokine secretion from visceral adipocytes

Recent studies suggest a relationship between intestinal microbiota and metabolic syndromes; however, the underlying mechanism remains unclear. To clarify this issue, we assessed the effects of bacterial cell wall components on adiponectin, leptin and resistin secretion from rat visceral adipocytes in vitro. We also measured the relative population of Firmicutes and Bacteroidetes in fecal microbiota and the amount of fecal mucin as an intestinal barrier function, when mice were fed a high-fat diet. In the present study, we demonstrated that bacterial cell wall components affect the secretion of adipokines, depending on the presence of antigens from gram-positive or gram-negative bacteria. Lipopolysaccharide markedly inhibited adiponectin, leptin, and resistin secretion, whereas peptidoglycan increased adiponectin secretion and decreased resistin secretion in vitro. In vivo experiments showed that the high-fat diet increased the population of Firmicutes and decreased that of Bacteroidetes. In contrast, the high-fat diet downregulated the stool output and fecal mucin content. These results demonstrate that bacterial cell wall components affect the onset of metabolic syndromes by mediating the secretion of adipokines from visceral adipose tissue. Furthermore, we believe that metabolic endotoxemia is not due to the increasing dominance of gram-negative bacteria, Bacteroidetes, but due to the depression of intestinal barrier function.

Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin.

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelilal nests.

Dietary polyphenols increase fecal mucin and immunoglobulin A and ameliorate the disturbance in gut microbiota caused by a high fat diet

The effects of dietary polyphenols on human health have mainly been discussed in the context of preventing degenerative diseases, particularly cardiovascular diseases and cancer. The antioxidant properties of polyphenols have been widely studied, but it has become clear that the mechanism of action of polyphenols extends beyond the modulation of oxidative stress, as they are poorly absorbed from the digestive tract. The purpose of this study was to clarify the effects of polyphenols on the colonic environment, intestinal barrier function, and gut microbiota. We demonstrated that dietary polyphenols derived from aronia, haskap, and bilberry, markedly elevated the amount of fecal mucin and immunoglobulin A (IgA) as an intestinal barrier function and ameliorated the disturbance in gut microbiota caused by a high fat diet in rats. These results suggest that dietary polyphenols play a significant role in the prevention of degenerative diseases through improvement of the colonic environment without any absorption from the digestive tract.

Cell Patterning Using a Template of Microstructured Organosilane Layer Fabricated by Vacuum Ultraviolet Light Lithography

Micropatterning techniques have become increasingly important in cellular biology. Cell patterning is achieved by various methods. Photolithography is one of the most popular methods, and several light sources (e.g., excimer lasers and mercury lamps) are used for that purpose. Vacuum ultraviolet (VUV) light that can be produced by an excimer lamp is advantageous for fabricating material patterns, since it can decompose organic materials directly and efficiently without photoresist or photosensitive materials. Despite the advantages, applications of VUV light to pattern biological materials are few. We have investigated cell patterning by using a template of a microstructured organosilane layer fabricated by VUV lithography. We first made a template of a microstructured organosilane layer by VUV lithography. Cell adhesive materials (poly(d-lysine) and polyethyleneimine) were chemically immobilized on the organosilane template, producing a cell adhesive material pattern. Primary rat cardiac and neuronal cells were successfully patterned by culturing them on the pattern substrate. Long-term culturing was attained for up to two weeks for cardiac cells and two months for cortex cells. We have discussed the reproducibility of cell patterning and made suggestions to improve it.

A New Method for in Vitro Calcification Using Acrylamide Gel and Bovine Serum

To investigate the mechanism of biological calcification in vitro, a model system consisting of an acrylamide gel block (1 x 3 x 3 mm) and fetal bovine serum was developed. Mineral deposition was induced in gel blocks which were immersed in 300 microliters of fetal bovine serum at 37 degrees C for 7 days in a CO2 incubator. X-ray diffraction indicated that the mineral was hydroxyapatite with low crystallinity. Effects of the concentration of acrylamide gel, the partial pressure of CO2 and matrix proteins within the gel on the mineral formation were investigated. In the gel concentration range of 10-60%, the largest amount of crystal grew in 40% acrylamide gel, where the serum protein did not penetrate. With an increase in the partial pressure of CO2 the Ca content in the gel block increased, reached the highest level at about 3.5% CO2 and then began to decrease. In 40% gel and at 5% CO2, the mineral formation was enhanced by phosvitin, phosphophoryn, demineralized dentin powder and alkaline phosphatase. Mineral deposition occurred around the collagen fibers immobilized in 40% acrylamide gel. These results indicate that 1) a putatively serum-derived inhibitor of calcification with high-molecular weight was prevented from penetrating into the 40% acrylamide gels, 2) immobilized polyanionic proteins and alkaline phosphatase were able to increase mineral deposition and 3) the partial pressure of CO2 greatly influenced the mineral deposition. It was concluded that this gel system is useful to investigate the mechanism of biological calcification in vitro.

Physiological levels of insulin and IGF‐1 synergistically enhance the differentiation of mesenteric adipocytes.

Visceral adipose tissue, particularly mesenteric adipose tissue, is important in the pathogenesis of metabolic syndrome. Here, we present a physiologically relevant differentiation system of rat mesenteric‐stromal vascular cells (mSVC) to mesenteric‐visceral adipocytes (mVAC). We optimized the insulin concentration at levels comparable to those in vivo (∼0.85 ng/ml) by including physiological concentrations of IGF‐1. We found that the insulin‐like growth factor (IGF‐1) and insulin worked synergistically, since IGF‐1 alone could induce CCAAT/enhancer binding protein alpha (C/EBPα) and adipocyte lipid binding protein (aP2) mRNA expression but not lipid droplet accumulation associated with maturation. Using real‐time PCR analyses on 180 adipocyte‐related genes, we identified a dramatic effect by IGF‐1 plus insulin. We also demonstrated the state of insulin resistance at pathologically high insulin concentrations. This culture system will contribute to understanding the physiological differentiation process and the patho/physiology of mVAC.

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.

Anthocyanin-rich Phytochemicals from Aronia Fruits Inhibit Visceral Fat Accumulation and Hyperglycemia in High-fat Diet-induced Dietary Obese Rats

Aronia fruits (chokeberry: Aronia melanocarpa E.) containing phenolic phytochemicals, such as cyanidin 3-glycosides and chlorogenic acid, have attracted considerable attention because of their potential human health benefits in humans including antioxidant activities and ability to improved vision. In the present study, the effects of anthocyanin-rich phytochemicals from aronia fruits (aronia phytochemicals) on visceral fat accumulation and fasting hyperglycemia were examined in rats fed a high-fat diet (Experiment 1). Total visceral fat mass was significantly lower in rats fed aronia phytochemicals than that in both the control group and bilberry phytochemicals-supplemented rats (p < 0.05). Moreover, perirenal and epididymal adipose tissue mass in rats fed aronia phytochemicals was significantly lower than that in both the control and bilberry phytochemicals group. Additionally, the mesenteric adipose tissue mass in aronia phytochemicals-fed rats was significantly low (p < 0.05). Furthermore, the fasting blood glucose levels significantly decreased in rats fed aronia phytochemicals for 4 weeks compared to that in the control rats (p < 0.05). Therefore, we investigated the effects of phytochemicals on postprandial hyperlipidemia after corn oil loading in rats, pancreatic lipase activity in vitro, and the plasma glycemic response after sucrose loading in order to elucidate the preventive factor of aronia phytochemical on visceral fat accumulation. In the oral corn oil tolerance tests (Experiment 2), aronia phytochemicals significantly inhibited the increases in plasma triglyceride levels, with a half-maximal inhibitory concentration (IC(50)) of 1.50 mg/mL. However, the inhibitory activity was similar to that of bilberry and tea catechins. In the sucrose tolerance tests (Experiment 3), aronia phytochemicals also significantly inhibited the increases in blood glucose levels that were observed in the control animals (p < 0.05). These results suggest that anthocyanin-rich phytochemicals in aronia fruits suppress visceral fat accumulation and hyperglycemia by inhibiting pancreatic lipase activity and/or intestinal lipid absorption.