Clostridium butyricum MIYAIRI 588 improves high-fat diet-induced non-alcoholic fatty liver disease in rats.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease (NAFLD) has become a common liver disease, as its prevalence has increased markedly in recent decades. The aim of the present study was to examine the improving effect of Clostridium butyricum MIYAIRI 588 (CBM588), a probiotic in clinical use for antibiotic-associated diarrhea, against high-fat diet (HFD)-induced fatty liver in rats. METHODS: After feeding HFD or HFD coated with CBM588 (HFD-CBM) for 12 weeks, we evaluated the hepatic mRNA levels related to lipid metabolism, and then assessed the hepatic protein levels of several transcription factors regulating these lipogenic gene expressions. RESULTS: The HFD-CBM group had decreased accumulation of lipid droplets in the liver compared with the HFD group. The HFD-CBM group had significantly decreased diacylglycerol acyltransferase (DGAT) 2 mRNA in the liver compared with the HFD group, whereas DGAT1 mRNA did not change between the HFD group and the HFD-CBM group. Moreover, the HFD-CBM group had significantly increased hepatic mRNA regulating cholesterol catabolism enzymes and excretion transporters. Correspondingly, the HFD-CBM588 groups had increased hepatic protein levels of peroxisome proliferator-activated receptor α/γ and liver X receptor α compared with the HFD group. The HFD-CBM group had accelerated excretion of total bile acid and non-esterified fatty acid in the feces. CONCLUSIONS: CBM588 intake may have novel potential for improving NAFLD.

Geometrical separation method for lipoproteins using bioformulated-fiber matrix electrophoresis: size of high-density lipoprotein does not reflect its density.

The increasing number of patients with metabolic syndrome is a critical global problem. In this study, we describe a novel geometrical electrophoretic separation method using a bioformulated-fiber matrix to analyze high-density lipoprotein (HDL) particles. HDL particles are generally considered to be a beneficial component of the cholesterol fraction. Conventional electrophoresis is widely used but is not necessarily suitable for analyzing HDL particles. Furthermore, a higher HDL density is generally believed to correlate with a smaller particle size. Here, we use a novel geometrical separation technique incorporating recently developed nanotechnology (Nata de Coco) to contradict this belief. A dyslipidemia patient given a 1-month treatment of fenofibrate showed an inverse relationship between HDL density and size. Direct microscopic observation and morphological observation of fractionated HDL particles confirmed a lack of relationship between particle density and size. This new technique may improve diagnostic accuracy and medical treatment for lipid related diseases.

A possible role of lysophospholipids produced by calcium-independent phospholipase A(2) in membrane-raft budding and fission.

Phospholipase A(2) (PLA(2)) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-beta-cyclodextrin. Activation of calcium-independent PLA(2) (iPLA(2)) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p<0.01), which was blocked by PLA(2) inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (<10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA(2) to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA(2) may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA(2) may provide a therapeutic target for viral infections.

Direct and simple fluorescence detection method for oxidized lipoproteins.

The quantification of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) is currently one of the most important clinical measurements for characterizing metabolic syndrome. However, recent studies have revealed additional factors that may be more strongly associated with the coronary heart disease than simple measurement of LDL or HDL levels, such as small dense (sd) LDL particles and oxidized LDL or HDL particles. Although several methods using enzyme-antibody detection systems or fluorescent probes have been devised to characterize these factors, such methods are expensive to implement for clinical measurements. Here, we present a straightforward analytical method for direct quantitation of oxidized lipoproteins by fluorescence spectrometry, with excitation in the UV (365 +/- 10 nm) or visible (470 +/- 10 nm) range and emission detected at 450 +/- 30 nm or 535 +/- 15 nm. This method can be readily applied for clinical measurement in patients with dyslipidemia using only 1 microL of 1 mg/mL of lipoprotein and without the need for any expensive detection antibodies. Using this new technique, biological samples from patients with dyslipidemia showed higher fluorescence intensities than samples from normal subjects when detecting oxidized LDL and light HDL (d = 1.063-1.125 g/mL), whereas samples from patients with dyslipidemia showed lower fluorescence intensities than samples from normal subjects when measuring oxidized heavy HDL (d = 1.125-1.210 g/mL) levels.

Lysophosphatidylcholine for efficient intestinal lipid absorption and lipoprotein secretion in caco-2 cells.

Phosphatidylcholine (PC) and its hydrolysates are considered to stimulate intestinal lipid absorption, however, their exact effects on lipoproteins and apolipoprotein (apo) metabolism remain ambiguous. This study aimed to further differentiate the effects of them using fully differentiated enterocyte-like Caco-2 cells. Lipid micelles (oleic acid 0.6, cholesterol 0.05, monooleylglycerol 0.2, taurocholate 2 in mmol/l) with or without choline, PC, and lysoPC (0.2 mmol/l each) were applied apically to Caco-2 cells. (3)H-oleic acid and (14)C-cholesterol were added to the micelles when necessary. Secreted lipoproteins were analyzed by a HPLC method. LysoPC had the most potent promoting effect on lipid uptake, and lipoprotein and apolipoprotein B-48 secretion among the molecules tested. LysoPC doubled the output of cholesterol and triglyceride as the lipoprotein component, but PC did not. On the other hand, PC only increased the secretion of apoA-IV in the presence of lipid micelles. These findings confirm that the alteration of PC by PLA(2) hydrolysis is intrinsically involved in the intestinal lipid absorption process and suggest that PC and its hydrolysis are coordinately associated with not only lipid absorption efficiency but also lipoprotein output and metabolism.

A small amount of tetrachloroethylene ingestion from drinking water accelerates antigen-stimulated allergic responses.

Previously, we observed that tetrachloroethylene (perchloroethylene, PCE) increased histamine release and inflammatory mediator production from antigen-stimulated mast cells. In this study, we examined the enhancing effect of low concentrations of PCE in drinking water on antigen-stimulated allergic responses. After exposure of Wistar rats to PCE in drinking water for 2 or 4 weeks, we performed a passive cutaneous anaphylaxis (PCA) reaction. PCE exposure for 4 weeks enhanced PCA reaction in a dose-dependent manner. In pathological studies, PCE exposure for 2 weeks exacerbated inflammation characterized by infiltration of lymphocytes and accumulation of mast cells around the vessel. Non-purified mast cells (NPMCs) from rats treated with 1mg/L PCE in drinking water for 2 weeks increased antigen-stimulated histamine release. Furthermore, the leukocytes of rats treated with PCE in drinking water for 4 weeks showed increased interleukin (IL)-4 expression. The mechanism of enhancing the PCA reaction is assumed to be that PCE increases IL-4 production and PCE causes T helper (Th) 1/Th2-type helper T-cell imbalance and increases histamine release from excessively accumulated mast cells. The results suggest that the intake of PCE in drinking water, even at a low concentration, leads to the initiation and acceleration of allergic diseases.

Enhancing effect of chlorinated organic solvents on histamine release and inflammatory mediator production.

We investigated the effect of several chlorinated organic solvents on antigen-induced histamine release and inflammatory mediator production. Non-purified rat peritoneal mast cells (NPMC) and rat basophilic leukemia (RBL-2H3) cells were sensitized with anti-dinitrophenol (DNP) monoclonal IgE antibody, and then stimulated with DNP-conjugated bovine serum albumin (DNP-BSA) and several chlorinated organic solvents. Trichloroethylene (TCE) and tetrachloroethylene (PCE) enhanced histamine release from antigen-stimulated NPMC and RBL-2H3 in a dose-dependent manner. In addition, TCE and PCE increased IL-4 and TNF-alpha production from antigen-stimulated RBL-2H3. In an in vivo study, we investigated the effect of TCE and PCE on passive cutaneous anaphylaxis (PCA) reaction. TCE and PCE enhanced PCA reaction markedly. These results suggest that TCE and PCE increase histamine release and inflammatory mediator production from antigen-stimulated mast cells via the modulation of immune responses. In addition, exposure to TCE and PCE may lead to the augmentation of allergic diseases.

Augmentation of antigen-stimulated allergic responses by a small amount of trichloroethylene ingestion from drinking water.

In previous report, we have shown that trichloroethylene (TCE) increases histamine release and inflammatory cytokine production from antigen-stimulated mast cells. In this study, we examined the enhancing effect of a small amount of TCE ingestion from drinking water on antigen-stimulated allergic responses. After exposure of Wistar rats to TCE ingestion for 2 or 4 weeks, we performed a passive cutaneous anaphylaxis (PCA) reaction. TCE ingestion for 2 and 4 weeks enhanced PCA reaction in a dose-dependent manner. On histological examination, TCE ingestion for 2 weeks exacerbated inflammation characterized by infiltration of lymphocytes and accumulation of mast cells around the vessel in the skin. After TCE ingestion for 4 weeks, the mesenteric lymph nodes (MLNs) showed increase of the size and wet weight, and germinal centers changed distinctly. The interleukin-4 (IL-4) mRNA levels on spleen, MLNs and leukocytes were increased. Moreover, serum total IgE levels of TCE ingestion increased in a time-dependent manner. Our results suggest that TCE ingestion induces pro-inflammatory responses and causes Th1/Th2-type helper T-cell imbalance. And more, a small amount of TCE ingestion may lead to the initiation and acceleration of type I allergic reaction.

Analysis of lipid raft molecules in the living brain slices.

Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices.

Effects of Catechins and Their Related Compounds on Cellular Accumulation and Efflux Transport of Mitoxantrone in Caco-2 Cell Monolayers.

The ability of catechins and their related compounds to inhibit breast cancer resistance protein (BCRP) function in Caco-2 cell monolayers was investigated with mitoxantrone as a BCRP substrate. The gallate or pyrogallol moiety on the catechin structure seemed to promote increased cellular accumulation and inhibit efflux transport of mitoxantrone. The ability of gallate catechins such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) to increase cellular accumulation and inhibit efflux transport of mitoxantrone was greater than that of nongallate catechins. Gallic acid octyl ester (GAO) also increased intracellular mitoxantrone accumulation. Experiments using GAO derivatives indicated that the gallate moiety required the presence of a long carbon chain for BCRP inhibition. Cellular accumulation and reduced efflux transport of mitoxantrone were greater with epigallocatechin 3-(3″-O-butyl) gallate than with EGCG. EGCG inhibition of BCRP seemed to be restricted by hydrophobicity. The co-administration of catechins, particularly EGCG and related compounds, with greater hydrophobicity may increase the therapeutic activities of BCRP substrates such as mitoxantrone.

Acarbose improves fibrinolytic activity in patients with impaired glucose tolerance.

Acarbose has been shown to ameliorate insulinemia, suggesting that it may exert favorable effects on the impaired fibrinolytic state in prediabetic patients. We therefore conducted a randomized controlled study to examine the effects of acarbose on fibrinolysis in patients with impaired glucose tolerance (IGT). The participants were randomized to receive (n = 20) or not (control, n = 20) 100 mg of acarbose before each meal (300 mg/d) for 3 months. A marked decrease in the plasma levels of plasminogen activator inhibitor 1 (by 42%) and fibrinogen (by 27%) was observed in the acarbose group at the end of the study, whereas no significant changes in the levels of these parameters were observed in the control group. We also conducted postprandial evaluation of insulin-related clinical markers and found ameliorated hyperinsulinemia in the subjects treated with acarbose. These results indicate that acarbose could improve fibrinolysis in patients with IGT, mainly by ameliorating insulinemia. Other favorable effects of acarbose, such as reduction in the plasma levels of oxidized low-density lipoprotein, glucose toxicity, and hyperglycemia, might also contribute, at least in part, to the beneficial effects of the drug on the fibrinolytic state in patients with IGT.

Dose calculation with a cone beam CT image in image-guided radiation therapy.

A kilo-voltage cone-beam CT (CBCT) attached to a linear accelerator can verify a target position in each radiation therapy. If CBCT images can be used in dose calculation, we can verify an actual dose distribution on every treatment day. However, the CBCT images are degraded by several factors, and so we cannot use the CBCT images directly in place of conventional multi-slice CT (MSCT) images that are used in the initial dose planning. In this paper, we proposed a new method for using CBCT and MSCT images in the calculation of a dose distribution. Our proposed method segments the CBCT and MSCT images into regions of three major organs (lungs, bones and soft tissues) by use of histogram analysis. We also calculated a value such as the median of the MSCT numbers in each region of the MSCT images, and we set three representative values to the corresponding regions of the CBCT images. In the calculation of a dose distribution, we used these modified CBCT images. The validity of our method was confirmed with experiments in which we used images of a heterogeneous phantom and patients' lungs in comparison with conventional methods. The results showed that the dose distribution determined by our method was similar to that of the initial dose plan, and our method was superior to the conventional methods in terms of pass rates of a distance-to-agreement analysis and γ analysis. The results of a dose-volume-histogram analysis also showed the accuracy of our proposed method.

Enhancing effects of trichloroethylene and tetrachloroethylene on type I allergic responses in mice.

Trichloroethylene (TCE) and tetrachloroethylene (perchloroethylene; PCE) are commonly identified as environmental contaminants of groundwater. Previously, we investigated the enhancing effects of TCE and PCE on antigen-induced histamine release and inflammatory mediator production in rat mast cells. In this study, to examine the potential effect of TCE and PCE on antigen-induced histamine release from mouse mast cells, mouse bone marrow-derived mast cells (BMMC) were sensitized with anti-dinitrophenol (DNP) monoclonal IgE antibody and then stimulated with DNP-BSA containing with TCE or PCE. Both TCE and PCE significantly enhanced antigen-induced histamine release from BMMC. Next we investigated the effects of TCE and PCE on the passive cutaneous anaphylaxis (PCA) reaction in vivo using ICR mice. TCE and PCE significantly enhanced the PCA reaction in a dose-dependent manner. In addition, we examined the enhancing effects of ingesting small amount of TCE and PCE in drinking water on antigen-stimulated allergic responses. After the ICR mice had ingested TCE or PCE in their drinking water for 2 or 4 weeks, we performed the PCA reaction. Both TCE and PCE ingestion enhanced the PCA reaction in a dose-dependent manner for 4 weeks. These results suggest that exposure to TCE and PCE leads to the augmentation of type I allergic responses in many species.

Enhancement of immediate allergic reactions by trichloroethylene ingestion via drinking water in mice.

The prevalence of allergic disorders is increasing in industrial areas and countries. Recent reports suggest that some environmental pollutants are related to the increase in allergic diseases, and we reported that trichloroethylene (TCE) is a candidate chemical for causing the increase of allergic diseases, as TCE ingestion is associated with allergic reaction enhancement. TCE is widely used in many industries, and it is commonly detected as an environmental contaminant. This study aimed to clarify the immunotoxicity of TCE in detail. BALB/c mice were treated with TCE dissolved in drinking water for 2 and 4 weeks, and the mice were immunized with ovalbumin (OVA)/aluminum hydroxide (alum) twice. On the final day of the TCE exposure period, we measured the active cutaneous anaphylaxis (ACA) reaction and the antigen- specific IgE level in serum as well as the histamine level at the allergic reaction site and assayed the proliferation rates of splenocytes collected from the animals. The ACA reaction was enhanced by TCE ingestion. The OVA specific IgE level in mice was enhanced by TCE exposure for 4 weeks. The proliferation rate of the splenocytes was enhanced by TCE ingestion for 2 and 4 weeks. The enhancement of the ACA reaction by TCE ingestion via drinking water may be related to the increase in splenocyte proliferation. On the other hand, it may be weakly related to antigen-specific IgE production.

A promoter in the novel exon of hPPARgamma directs the circadian expression of PPARgamma.

AIM: PPARgamma (peroxisome proliferator-activated receptor gamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. Herein, we show that expression levels of the novel PPARgamma transcript exhibit circadian oscillation. To study the mechanisms controlling PPARgamma expression, a novel PPARgamma gene promoter was cloned and characterized. METHODS: We analyzed the novel PPARgamma promoter by luciferase reporter assays and gel shift analysis. RESULTS: Surprisingly, it was not an intron but rather the novel first exon of PPARgamma that was found to have functional minimal promoter activity. Luciferase reporter assays and gel shift assays revealed that the novel first exon is essential for novel PPARgamma promoter activation and that DBP (albumin gene D-site binding protein) and E4BP4 (E4 promoter A binding protein 4) bind directly to D-sites in the novel first exon. CONCLUSION: Our results demonstrate that the PAR-bZIP (bZIP, basic leucine zipper) family and E4BP4 are the main regulatory factors involved in oscillation of novel PPARgamma expression. This regulatory mechanism clearly differs from that of the circadian expression of PPARalpha.

Activating effect of momordin, extract of bitter melon (Momordica Charantia L.), on the promoter of human PPARdelta.

AIM: Bitter melon (Momordica charantia L.) is a common vegetable grown in Okinawa that has also been used recently in medicine for the treatment of diseases such as diabetes, hypertension, and dyslipidemia. Among Bitter melon extracts compounds, we focused on an extract known as momordin in the present study, to examine its effect on peroxisome-proliferator activated-receptor (PPAR) delta (also called PPARdelta in rodents) expression and promoter activity of the human PPARdelta gene. METHODS: A human PPARdelta promoter-reporter plasmid was made as a template from a BAC CLONE (RPCI-11C) containing a -3076 bp (BglI site) +74 bp (EcoRI site) sequence. Luciferase assay of PPARdelta promoter activity was performed using HepG2 cells. RESULTS: 10 and 25 nM Momordin significantly increased the expression of PPARdelta mRNA 1.5-fold (relative to the control). Moreover, 10 and 25 nM Momordin significantly increased PPARdelta promoter activity in a dose-dependent manner, reaching more than 1.5-fold relative to the control. CONCLUSION: Our present data obtained through successful cloning of the PPARdelta promoter demonstrate that PPARdelta production and activation are upregulated through PPARdelta promoter activity following momordin treatment.

Rapid and simple profiling of lipoproteins by polyacrylamide-gel disc electrophoresis to determine the heterogeneity of low-density lipoproteins (LDLs) including small, dense LDL.

This study aimed to explore the potential of polyacrylamide-gel disc electrophoresis (PAGE) for lipoprotein profiling in clinical practice. Blood samples were collected from 146 patients with type 2 diabetes mellitus and lipid parameters were assayed by PAGE, including small, dense low-density lipoprotein (LDL) (n = 41), and triglyceride-rich lipoprotein remnant cholesterol (n = 37). We also used a commercial kit to measure small, dense LDL (n = 41). By PAGE, we obtained the percentage of the area under the curve (AUC %) of each peaks and calculated respective AUC% x total cholesterol (AUC%xTC) values. The calculated values of LDL-AUC%xTC, small LDL-AUC%xTC, and HDL-AUC%xTC values were correlated well with values from homogeneous assay for LDL-cholesterol, small, dense LDL-cholesterol, and HDL-cholesterol assays (r = 0.94, 0.81, and 0.89, respectively). PAGE combined with measurement of total cholesterol and triglycerides provides a rapid evaluation of anti- or pro-atherogenic lipoproteins and a simple profiling system for both the "quantity" and "quality" of lipoproteins, allowing a better assessment of the risk of coronary artery diseases. This article discusses several methods for simple and rapid lipid profiling and outlines some recent patents relevant to the methods.

Statins Activate Human PPARalpha Promoter and Increase PPARalpha mRNA Expression and Activation in HepG2 Cells.

Statins increase peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA expression, but the mechanism of this increased PPARalpha production remains elusive. To examine the regulation of PPARalpha production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) on human PPARalpha promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1) Majority of statins enhanced PPARalpha promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPARalpha promoter. This enhancement may be mediated by statin-induced HNF-4alpha. (2) PPARalpha mRNA expression was increased by statin treatment. (3) The PPARalpha levels in nuclear fractions were increased by statin treatment. (4) Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPARalpha/RXRalpha expression vectors. In summary, these data demonstrate that PPARalpha production and activation are upregulated through the PPARalpha promoter activity by statin treatment.

Disruption of the murine intestinal alkaline phosphatase gene Akp3 impairs lipid transcytosis and induces visceral fat accumulation and hepatic steatosis.

Intestinal alkaline phosphatase (IAP) is involved in the process of fat absorption, a conclusion confirmed by an altered lipid transport and a faster body weight gain from 10 to 30 wk in both male and female mice with a homozygous null mutation of the IAP coding gene (Akp3(-/-) mice). This study was aimed to delineate morphologically and quantitatively the accelerated lipid absorption in male Akp3(-/-) mice. Feeding a corn oil bolus produced an earlier peak of triacylglycerol in serum (2 vs. 4 h for Akp3(-/-) and wild-type mice, respectively) and an approximately twofold increase in serum triacylglycerol concentration in Akp3(-/-) mice injected with a lipolysis inhibitor, Triton WR-1339. A corn oil load induced the threefold enlargement of the Golgi vacuoles in male wild-type mice but not in Akp3(-/-) mice, indicating that absorbed lipids rarely reached the Golgi complex and that the transcytosis of lipid droplets does not follow the normal pathway in male Akp3(-/-) mice. Force feeding an exaggerated fat intake by a 30% fat chow for 10 wk induced obesity in both male Akp3(-/-) and wild-type mice, and therefore no phenotypic difference was observed between the two. On the other hand, the forced high-fat chow induced an 18% greater body weight gain, hepatic steatosis, and visceral fat accumulation in female Akp3(-/-) mice but not in female wild-type controls. These results provide further evidence that IAP is involved in the regulation of the lipid absorption process and that its absence leads to progressive metabolic abnormalities in certain fat-forced conditions.