Extracellular vesicles of Weizmannia coagulans lilac-01 reduced cell death of primary microglia and increased mitochondrial content in dermal fibroblasts in vitro.

We investigated the properties of extracellular vesicles from the probiotic Weizmannia coagulans lilac-01 (Lilac-01EVs). The phospholipids in the Lilac-01EV membrane were phosphatidylglycerol and mitochondria-specific cardiolipin. We found that applying Lilac-01EVs to primary rat microglia in vitro resulted in a reduction in primary microglial cell death (P < .05). Lilac-01EVs, which contain cardiolipin and phosphatidylglycerol, may have the potential to inhibit cell death in primary microglia. The addition of Lilac-01EVs to senescent human dermal fibroblasts suggested that Lilac-01 EVs increase the mitochondrial content without affecting their membrane potential in these cells.

Cloaking the ACE2 receptor with salivary cationic proteins inhibits SARS-CoV-2 entry.

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

A simple culture method for liver and intestinal tissue-resident macrophages from neonatal mice.

The liver and intestine contain a remarkably large portion of tissue-resident macrophage cells representing a phenotype that downregulates inflammation and initiates tissue repair. Here, liver and intestinal tissues obtained from neonatal mice were minced, enzymatically digested, and incubated in RPMI1640-based media. In a 2-wk culture, spherical floating cells emerged on a fibroblastic sheet. These cells showed phagocytic activity and F4/80+-CD11b+-CD206+-Arg1+-iNOS--CD209a- phenotype, suggesting that these cells are tissue-resident macrophages. These macrophages proliferated in the co-culture system in the presence of fibroblastic feeder cell layer and absence of supplemental cytokines; the co-culture system did not cause a significant change in the phenotype of cells grown in a 4-wk culture. On the feeder cells, macrophage density was approximately 1.5 × 104/cm2 and the doubling time was approximately 70 h. Based on these observations, we present a simple method for the isolation and propagation of tissue-resident macrophages resembling M2 macrophage from neonatal mice, and this method provides a useful platform for in vitro studies of tissue-resident macrophages.

Effects of 4-week continuous ingestion of champignon extract on halitosis and body and fecal odor.

This was placebo-controlled double-blind parallel-group comparative clinical trial targeting 80 men and women aged 50-79 years with halitosis and body and fecal odor. We investigated whether daily champignon extract ingestion for 4 weeks improved these conditions. Subjects were divided into four groups: a placebo group and 50, 500, and 1000 mg/day ingestion groups. No severe adverse events or side effects were noted during the study period. Compared with the placebo group, all champignon extract ingestion groups showed improvement or tendency toward improvement in halitosis and body and fecal odor. Furthermore, our results suggested that the effectiveness of champignon extract in alleviating odors is dose-dependent, i.e., it increases with the dosage.

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.

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.

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.

Control of homeostatic and pathogenic balance in adipose tissue by ganglioside GM3.

Ganglioside GM3 (Siaα2-3Galß1-4Glcß1-1Cer) has been known to participate in insulin signaling by regulating the association of the insulin receptor in caveolae microdomains (lipid rafts), which is essential for the execution of the complete insulin metabolic signaling in adipocytes. Macrophage-secreted factors including proinflammatory cytokines, tumor necrosis factor-α and interleukin-1ß, in adipose tissues have been known to limit the local adipogenesis and induce insulin resistance; however, the interplay between adipocytes and macrophages upon regulation of GM3 expression is not clear. GM3 was virtually absent in primary adipocytes differentiated from macrophage-depleted mesenteric stromal vesicular cells, which accompanies enhancement of insulin signaling and adipogenesis. We found that the expression of GM3 is governed by soluble factors including steady-state levels of proinflammatory cytokines secreted from resident macrophages. The direct involvement of GM3 in insulin signaling is demonstrated by the fact that embryonic fibroblasts obtained from GM3 synthase (GM3S)-deficient mice have increased insulin signaling, when compared with wild-type embryonic fibroblasts, which in turn leads to enhanced adipogeneis. In addition, GM3 expression in primary adipocytes is increased under proinflammatory conditions as well as in adipose tissue of diet-induced obese mice. Moreover, GM3S-deficient mice fed high-fat diets become obese but are resistant to the development of insulin resistance and chronic low-grade inflammatory states. Thus, GM3 functions as a physiological regulatory factor of the balance between homeostatic and pathological states in adipocytes by modulating insulin signaling in lipid rafts.

Double network hydrogels from polyzwitterions: high mechanical strength and excellent anti-biofouling properties.

Polyzwitterionic materials, which have both cationic and anionic groups in the polymeric repeat unit, show excellent anti-biofouling properties and are drawing more attention in the biomedical field. In this study, we have successfully synthesized novel single network hydrogels and double network (DN) hydrogels from the zwitterionic monomer, N-(carboxymethyl)-N,N-dimethyl-2-(methacryloyloxy) ethanaminium, inner salt (CDME). The polyCDME (PCDME) single network hydrogel behaves like a hydrophilic neutral hydrogel and its properties are not sensitive to temperature, pH, or ionic strength over a wide range. DN hydrogels using the poly(2-acrylamido-2-methylpropanesulfonic) (PAMPS) as the first network and PCDME as the second network, having a Young's modulus of 0.2-0.9 MPa, possess excellent mechanical strength (fracture stress: 1.2-1.4 MPa, fracture strain: 2.2-6.0 mm/mm) and toughness (work of extension at fracture: 0.9-2.4 MJ m-3) depending on the composition ratio of PCDME to PAMPS. The strength and toughness of the optimized PAMPS/PCDME DN is comparable to the normal PAMPS/PAAm DN hydrogels that use poly(acrylamide) (PAAm) as the second network. By macrophage adhesion test, both the PCDME hydrogels and the PAMPS/PCDME DN hydrogels have shown excellent anti-biofouling properties. These results demonstrate that the PCDME-based DN hydrogels have high potential as a novel soft and wet biomaterial.

Optimal temperature range for low-temperature preservation of dissociated neonatal rat cardiomyocytes.

The increase in demand for primary cardiomyocytes necessitates advanced methods for their stable supply. In this study, we investigated the optimal temperature range for preserving dissociated cardiomyocytes for 72 h while maintaining their normal growth and beating functions. Neonatal rat cardiomyocytes dissociated by collagenase and suspended in the culture medium were preserved at temperatures from -2 to 35°C for 72 h. The cardiomyocytes preserved at temperatures below 20°C maintained the initial dispersed states, whereas they aggregated robustly at higher temperatures. The viability of the dispersed cells after preservation was more than 80%. After the preservation, the microscopic observations during the 7-days cultivation indicated that these dispersed cardiomyocytes grew normally to form a confluent monolayer, and beat spontaneously and regularly during culture, as did the fresh cells. These systematic evaluations indicated that the optimal temperature ranged from 3 to 20°C. Below this optimal temperature range, the cell activities decreased slightly with temperature. The robustly aggregated cardiomyocytes exhibited weak growth and low beating rates, although some cardiomyocytes still survived. The optimal conditions, which consist of a wider temperature range and longer preservation period than the present commercially used conditions, allowed milder temperature control and thus more economical transportation for the dissociated primary cardiomyocytes.

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.

Shrinking and development of lipid droplets in adipocytes during catecholamine-induced lipolysis.

Time-lapse observation of adipocytes during catecholamine-induced lipolysis clearly shows that shrinking of existing lipid droplets (LDs) occurs in some adipocytes and that small LDs are newly developed in almost all cells. Immunofluorescence imaging reveals that activation and localization of hormone-sensitive lipase (HSL) on the surface of LDs, which are required for conferring maximal lipolysis, are necessary for the shrinking of the LDs. However, not all adipocytes in which phosphorylated HSL is localized on LDs exhibit shrinking of LDs. The simultaneous shrinking and development of LDs yield apparent fragmentation and dispersion of LDs in adipocytes stimulated with catecholamine.

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.

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 ( approximately 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/EBPalpha) 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.

Regulation of the susceptibility to oxidative stress by cysteine availability in pancreatic beta-cells.

Pancreatic beta-cells are susceptible to oxidative stress, which is related closely to the islet dysfunction. In the present study, using the pancreatic cell lines HIT-T15 and RINm5F as known in vitro models of impaired beta-cell function as well as primary rat islet beta-cells, we observed a relationship between intracellular glutathione levels and oxidative stress-mediated cell dysfunction. Hydrogen peroxide and 4-hydroxy-2-nonenal caused cell death in HIT-T15 and RINm5F cells at lower concentrations compared with non-beta-cells, such as HepG2 and NRK-49F cells. The extent of the cytotoxicity caused by the model oxidants was inversely correlated well with intracellular glutathione levels in the cell lines used. Treatment of HIT-T15 and RINm5F cells with l-cysteine or l-cystine significantly augmented the glutathione contents, surpassing the effect of N-acetylcysteine, and abrogated 4-hydroxy-2-nonenal-mediated cytotoxicity almost completely. l-Cysteine increased intracellular glutathione levels in primary beta-cells as well. Supplementation of l-cysteine to the RINm5F cell culture inhibited 4-hydroxy-2-nonenal-mediated cytosolic translocation of PDX-1, a key transcription factor for beta-cell function. Intrinsic transport activities (V(max)/K(m)) of the l-cystine/l-glutamate exchanger in HIT-T15 and RINm5F cells were considerably lower than that in NRK-49F cells, although gene expressions of the exchanger were similar in these cells. Results obtained from the present study suggest that the restricted activity of the l-cystine/l-glutamate exchanger controls the levels of intracellular glutathione, thereby making beta-cells become susceptible to oxidative stress.

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-gamma 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 microM 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-gamma agonists on adipose tissues.

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 Dulbecco's modified Eagle medium (DMEM)/F12 containing 17 microM pantothenic acid, 33 microM biotin, 100 microM ascorbic acid, 1 microM octanoic acid, 50 nM triiodothyronine, 10 microg/ml insulin, 10% newborn calf serum (NCS), 100 units/ml penicillin and 100 microg/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)-gamma agonists. The SVCs differentiated and proliferated efficiently, and formed a confluent monolayer in 3 days. The VAs accumulated lipids droplets in their cytoplasm at approximately 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 x 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.

Endotoxin contamination contributes to the in vitro cytokine-inducing activity of osteopontin preparations.

Recently, both native and recombinant preparations of human osteopontin (OPN) have been shown to be able to induce the production of several proinflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) or purified monocytes. In the present study, we found that commercially available native and recombinant OPNs contain variable amounts of endotoxin (LPS) and that removal of endotoxin by polymyxin B-agarose column abrogated their cytokine-inducing activity. These results suggest the questionable evidence of the ability of OPN to induce several cytokines in human PBMCs and draw attention to the exquisite sensitivity of PBMCs/monocytes to endotoxin contaminants.