Lipid Profiling of Serum and Lipoprotein Fractions in Response to Pitavastatin Using an Animal Model of Familial Hypercholesterolemia.

Statins are widely used for the treatment of atherosclerotic cardiovascular diseases. They inhibit cholesterol biosynthesis in the liver and cause pleiotropic effects, including anti-inflammatory and antioxidant effects. To develop novel therapeutic drugs, the effect of blood-borne lipid molecules on the pleiotropic effects of statins must be elucidated. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for hypercholesterolemia, are suitable for the determination of lipid molecules in the blood in response to statins because their lipoprotein metabolism is similar to that of humans. Herein, lipid molecules were investigated by lipidome analysis in response to pitavastatin using WHHLMI rabbits. Various lipid molecules in the blood were measured using a supercritical fluid chromatography triple quadrupole mass spectrometry. Cholesterol and cholesterol ester blood concentrations decreased by reducing the secretion of very low density lipoproteins from the liver. Independent of the inhibition effects of cholesterol biosynthesis, the concentrations of some lipids with anti-inflammation and antioxidant effects (phospholipid molecules with n-6 fatty acid side chains, lysophosphatidylcholines, phosphatidylethanolamine plasmalogens, and ceramide molecules) were significantly altered. These findings may lead to further investigation of the mechanism of statin action.

Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry.

Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.-Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba.

Ameliorating effects of D-47, a newly developed compound, on lipid metabolism in an animal model of familial hypercholesterolemia (WHHLMI rabbits).

Improvements induced in lipid metabolism in the liver by D-47, a newly developed compound, were examined herein. WHHLMI rabbits, an animal model of hypercholesterolemia and coronary atherosclerosis, was fed D-47-supplemented chow for 5 weeks at a dose of 30mg/kg. Lipid concentration were assayed using enzymatic methods. Plasma lipoproteins were fractionated with an ultracentrifuge. mRNA expression was analyzed with real-time PCR. Lipidome analyses of lipoproteins were performed using supercritical fluid chromatography mass spectrometry. In the D-47-treated group, serum lipid levels decreased by 23% for total cholesterol and by 40% for triglycerides. These reductions were mainly attributed to decreases in the VLDL fraction. Compared with the control, in the D-47 group, lipid contents in the liver were decreased by 22% in cholesterol and by 69% in triglycerides, and fat accumulation was decreased by 57% in pericardial fat and by 17% in mesenteric fat. In lipidome analyses of VLDL fraction, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylethanolamine plasmalogen, sphingomyelin, and ceramide were decreased by the D-47 treatment. mRNA expression in the liver was 51% lower for FAS and 24% lower for MTP, but 5.9- and 5.1-fold higher for CYP7A1 and CPT-1, respectively, in the D-47 group than in the control. mRNA expression was 72%, 64%, and 36% higher for LPL, CTP-1, and PPARγ, respectively, in mesenteric fat in the D-47 group. D-47 is a potent lipid-lowering compound that uses a different mechanism of action from that of statins. It has potential as a compound in the treatment of steatohepatitis and metabolic syndrome.

The Coronary Artery Running Pattern is One of the Causes of Individual Differences in the Progression of Coronary Atherosclerosis in WHHLMI Rabbits, an Animal Model for Coronary Atherosclerosis.

AIMS: The relationship between the coronary artery running pattern and development of coronary lesions was re-examined herein using WHHLMI rabbits, an animal model of spontaneous coronary atherosclerosis. METHODS: The coronary artery running pattern was analyzed using an X-ray computed tomography (CT) apparatus after perfusion. Pathological sections were prepared (Victoria blue-HE staining) at 100 µm intervals from the origin of the left circumflex artery (LCX). The severity of coronary lesions was evaluated based on cross-sectional narrowing (lesion area/inner area of the internal elastic lamina). RESULTS: In the CT analysis, the angle of the main curvature of the LCX negatively correlated with the percentage of sections with lesions and cross-sectional narrowing. The percentage of sections with lesions was significantly higher in acute angle-type LCX than in obtuse angle-type LCX. Cross-sectional narrowing was also significantly greater in acute angle-type LCX than in obtuse angle-type LCX. The percentage of fibrous lesions was high at the proximal region of LCX, whereas that of lipid-rich lesions was high at the curvature. In 24 months age group, the percentage of sections with calcification in acute angle-type LCX was about twice that in obtuse angle-type LCX. CONCLUSIONS: Individual differences were observed in the angle of the main curvature of the LCX, which affected the occurrence and extension of atherosclerotic lesions.

Effects of Enzymatically Synthesized Glycogen and Exercise on Abdominal Fat Accumulation in High-Fat Diet-Fed Mice.

The combination of diet and exercise is the first choice for the treatment of obesity and metabolic syndrome. We previously reported that enzymatically synthesized glycogen (ESG) suppresses abdominal fat accumulation in obese rats. However, the effect of the combination of ESG and exercise on abdominal fat accumulation has not yet been investigated. Our goal in this study was therefore to evaluate the effects of dietary ESG and its combination with exercise on abdominal fat accumulation in high-fat diet (HFD)-fed mice. Male ICR mice were assigned to four groups: HFD, HFD containing 20% ESG, HFD with exercise, HFD containing 20% ESG with exercise. Treadmill exercise was performed for 3 wk (25 m/min, 30 min/d, 3 d/wk) after 5-d adaption to running at that speed. Both ESG and exercise significantly reduced the weights of abdominal adipose tissues. In addition, the combination of ESG and exercise significantly suppressed abdominal fat accumulation, suggesting that ESG and exercise showed an additive effect. Exercise significantly increased the mRNA levels of lipid metabolism-related genes such as lipoprotein lipase, peroxisome proliferator-activated receptor delta; factor-delta (PPARδ), carnitin palmitoyltransferase b, adipose triglyceride lipase (ATGL), and uncoupling protein-3 in the gastrocnemius muscle. On the other hand, dietary ESG significantly decreased the mRNA levels of PPARδ and ATGL in the gastrocnemius muscle. These results suggest that the combined treatment of ESG and exercise effectively suppresses abdominal fat accumulation in HFD-fed mice by different mechanisms.

Effects of dietary heme iron and exercise training on abdominal fat accumulation and lipid metabolism in high-fat diet-fed mice.

Animal by-products can be recycled and used as sources of essential nutrients. Water-soluble heme iron (WSHI), a functional food additive for supplementing iron, is produced by processing animal blood. In this study, we investigated the effects of dietary supplementation of 3% WSHI and exercise training for 4 weeks on the accumulation of abdominal fat and lipid metabolism in mice fed high-fat diet. Exercise-trained mice had significantly less perirenal adipose tissue, whereas WSHI-fed mice tended to have less epididymal adipose tissue. In addition, total weight of abdominal adipose tissues was significantly decreased in the Exercise + WSHI group. Dietary WSHI significantly increased the messenger RNA (mRNA) levels of lipoprotein lipase and hormone-sensitive lipase. WSHI-fed mice also tended to show increased mRNA levels of adipose triglyceride lipase in their epididymal adipose tissue. Dietary WSHI also significantly decreased the mRNA levels of fatty acid oxidation-related enzymes in the liver, but did not influence levels in the Gastrocnemius muscle. Exercise training did not influence the mRNA levels of lipid metabolism-related enzymes in the epididymal adipose tissue, liver or the Gastrocnemius muscle. These findings suggest that the accumulation of abdominal fat can be efficiently decreased by the combination of dietary WSHI and exercise training in mice fed high-fat diet.

[Effects of oral streptococci on biofilm formation by cariogenic bacteria in dual species cultures].

The effects of oral commensal streptococci (Streptococcus sanguinis, Streptococcus gordonii, Streptococcus mitis, and Streptococcus salivarius) on biofilm formation by cariogenic mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) were investigated. Cell suspensions were cultured on 96-well microtiter plates coated with or without salivary components (SC), and in flow cell systems coated with SC in tryptic soy broth including 0.25% sucrose without dextrose (TSB). The resultant biofilm formations were stained using safranin or a LIVE/DEAD BacLight Viability Kit, and examined with absorbance at 492 nm or using confocal laser scanning microscopy. Mutans streptococci and S. sanguinis biofilms were formed significantly on the polystyrene surfaces in TSB. Further, in combination cultures, S. sanguinis formed a sufficient biofilm when cultured with S. mutans. However, when S. sanguinis was cultured with S. sobrinus, biofilm formation was slightly inhibited. S. gordonii also inhibited biofilm formation in the culture with S. sobrinus, but not when cultured with S. mutans. S. mitis and S. salivarius collapsed the biofilm morphology and inhibited volume development in some conditions when cultured with S. mutans or S. sobrinus. Biofilm formation by mutans streptococci was challenged and collapsed on the whole by culturing with each of the other oral streptococci. These results indicate that co-culturing of multiple species of mutans streptococci and other oral streptococci has physical effects related to previous attachment and colonization on the surface, as well as biological effects to regulate biofilm formation.