In reviewing the trends and influences of life-style in this country on health and disease in the latter half of 20th century, we focused our attention on 4 major habits of smoking, drinking, exercise and diets, and collected data on the Japanese to conduct a meta-analysis of their relationship with serum lipids and lipoproteins, which are the metabolic risk factors most closely related to atherosclerosis. 1) The percentage of smokers was 54.0% in adult males and 14.5% in adult females in 1999. In the data of 7, 256 subjects (mean age 47 years) in 16 papers, smoking increased triglycerides by 13 mg/dl (0.15 mmol/L) or in 559 non-drinkers with a mean age of 49 years in 3 papers by 18 mg/dl (0.20 mmol/L), and decreased HDL-cholesterol by 3.5 mg/dl (0.09 mmol/L) with every 20 cigarettes smoked according to the regression equation. 2) As for drinking, the annual ethanol consumption per adult was 8.5L in 1996. The effects of alcohol on serum lipids were analyzed in 27, 035 males (mean age 47 years) in 24 studies. Drinking elevated triglycerides by a mean of 10 mg/dl (0.11 mmol/L), and also HDL-cholesterol by 2.5 mg/dl (0.06 mmol/L) per 23 g of alcohol intake (corresponding to 1 go of sake or 1 large bottle of beer). 3) Concerning exercise habit, 25% of males and 21% of females (mean age 47 years) regularly performed exercise such as jogging, swimming, aerobics, and tennis. However, walking was regarded as an easy exercise to be practiced by subjects of all ages. The effects of walking on serum lipids were studied in a total of 46, 074 subjects (mean age 47 years) in 8 populations. Triglycerides were significantly lower by 10 mg/dl (0.11 mol/L), and HDL-cholesterol higher by 3 mg/dl (0.08 mmol/L) in those who walked 6, 000 or more steps/day than in those who walked less than 2, 000 steps/day. The effects of harder exercise like jogging or swimming were analyzed in 2, 242 subjects in 14 papers (mean age 44 years). Triglycerides decreased by 10 mg/dl (0.11 mmol/L), and HDL-cholesterol elevated by 5 mg/di (0.13 mmol/L) with an increase in the exercise intensity by one level of about 300 kcal. In exercise therapy, triglycerides were decreased by a mean of 20 mg/dl (0.23 mmol/L), and HDL-cholesterol increased by a mean of 10 mg/dl (0.26 mmol/L) by exercise at a mean heart rate of about 135 bpm, which is equivalent to 50% VO2max for 30 minutes x 3 times/week. 4) In nutritional trends, the mean energy intake in 52 postwar years averaged 2, 116-F84 kcal with no marked changes according to nutritional surveys. However, the percentage of fat in total energy intake was lowest at 7% in 1946, increased thereafter until it exceeded 20% in 1973, and surpassed 25% in 1988. The mean total cholesterol level of the Japanese increased by 28 mg/di (0.72 mmol/L) in the past 30 years and reached 204 mg/di (5.28 mmol/L) in a survey in 1990. 5) Concerning dietary habits, total cholesterol was lower by a mean of 13 mg/di (0.34 mmol/L), triglycerides lower by 40 mg/di (0.45 mmol/L), and HDL-cholesterol higher by 5 mg/dl (0.13 mmol/L) in the group who ate 7 or more Japanese-style meals in the 9 meals during 3 days than in the group who ate 3 or less Japanese-style meals in the 9 meals. When serum lipids were compared among individuals living in cities (8 groups ; 3, 613 subjects ; mean age 51 years), agricultural villages (13 groups ; 5, 364 subjects ; mean age 51 years), and fishing villages (9 groups ; 1, 071 subjects ; mean age 52 years). Total cholesterol was lower by a mean of 10 mg/dl (0.26 mmol/L) in fishing villages than in cities, and triglycerides lower by a mean of 15 mg/dl (0.17 mmol/L) in fishing villages than in cities and agricultural villages. HDL-cholesterol was 5 mg/dl (0.13 mmol/L) higher in agricultural villages and 3 mg/dl (0.08 mmol/L) higher in fishing villages than in cities. 6) The effects of dietary therapy or guidance were evaluated in 585 subjects (mean age, 53 years) in 12 papers.

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The effects of bezafibrate treatment on lipoprotein metabolism were investigated in hypertriglyceridemic subjects. Bezafibrate, a fibric acid derivative, was administered at 200-400 mg/day to 8 patients with hyperlipoproteinemia (type lib and IV) for 3-6 months. We evaluated the effects of bezafibrate on the plasma levels of total cholesterol (chol), triglyceride (TG), and apoB. In addition, the lipid and apoB contents were also analyzed in VLDL, IDL, LDL and HDL fractions before and after the treatment. It was revealed that plasma levels of chol, TG and apoB significantly decreased after the treatment, 236.3 vs 210.9, 192.4 vs 90.2 (p< 0.01) and 129.8 vs 116.2 (p< 0.05) mg/dI respectively. VLDL-chol, VLDL-TG and VLDL-apoB dropped from 26.5, 127.6 and 11.1 mg/di to 9.1, 49.5 and 6.7 mg/ dl respectively after the treatment. Regarding qualitative alterations of VLDL, TG/apoB, chol/apoB and TG + chol/apoB ratios in VLDL were significantly reduced, indicating that the size of VLDL was diminished by the treatment. In addition, HDL-chol increased from 40.4 to 60.8 mg/di after the treatment. Consequently LDL-chol/HDL-chol significantly decreased. In conclusion, bezafibrate administration decreased the TG, chol and apoB content in VLDL, suggesting a reduced number of VLDL. Significant rise of HDL-chol and decrease of LDL-chol/HDL-chol are additional beneficial effects following bezafibrate treatment.

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HB₂ a candidate HDL receptor, is quite distinct from other HDL receptors in its structure. However, while changes in cellular cholesterol content, or a reduction in cholesterol biosynthesis accompany corresponding changes in HB₂ expression, the level at which these changes occur have not been determined and the regulation and the function of HB₂ remain uncertain. In order to further investigate the regulation of HB₂, we administered simvastatin to rabbits to reduce cholesterol biosynthesis and follow changes in HB₂ mRNA in various tissues. Six rabbits were given 15 mg/kg of simvastatin by oral administration daily and another six rabbits were given the same volume of saline as a control, for 21 days. They were then sacrificed to obtain samples of blood, liver, lung, jejunum and brain. Simvastatin reduced plasma total cholesterol by 47% and free cholesterol concentrations in liver and lung by 25 and 10%, respectively. Northern blot analysis showed that simvastatin lowered the expression of HB₂ significantly in the liver and lung by 54% and 42% respectively but not in the jejunum or brain. These results support the findings of a previous study showing that HDL binding activity of both HB₁ and HB₂, which was determined by ligand blotting using HDL₃ as a ligand, were reduced after administering cholesterol lowering agents. (Arteriosclerosis, 10 : 1045-1050, 1990). The present study suggests that simvastatin down-regulated HB₂ at the transcriptional stage. Although the complete physiological function of HB₂ is unclear, it appears to play some role in the cholesterol metabolism, warranting further studies to elucidate the nature of this interaction.

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We examined whether or not hydrogen peroxide induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by a propidium iodide assay. Under serum free conditions, treatment of the endothelial cells with hydrogen peroxide (H₂O₂) for 6 hours induced cytotoxicity (⁵¹Cr release) in a dose-dependent manner (10 μmol/l-1 mmol/l). Under the condition containing 10% serum, H₂O₂ did not induce cytotoxicity even at the highest concentration (1 mmol/l). However, concomitant treatment of endothelial cells with cycloheximide at a dose of 10 ug/ml elicited endothelial cell apoptosis of by 15.6±1.7% at 6 hours after administration, even under the 10% serum condition. In addition, endothelial cell apoptosis due to H₂O₂ and cycloheximide was completely inhibited by zD-dcb (50 μmol/l), an inhibitor of caspase. 1 mmol/l of 4, 4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS), which is a chloride bicarbonate exchanger blocker, partially inhibited the H₂O₂ and cycloheximide-induced endothelial cell apoptosis. On the other hand, cytotoxicity of endothelial cells due to H₂O₂ under serum free conditions was not inhibited by DIDS. These data suggested that hydrogen peroxide could induce endothelial cell apoptosis or cell membrane injury (⁵¹Cr release) in the presence or absence of an inhibitor of protein synthesis.

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Marine animals produce astaxanthin which is a carotenoid and antioxidant. In this study we determined the in vitro and ex vivo effects of astaxanthin on LDL oxidation. The oxidation of LDL was measured in a 1 ml reaction system consisting of increasing concentrations of astaxanthin (12.5, 25.0, 50.0 μg/ml), 400 μM V-70 (2, 2'-azobis (4-methoxy-2, 4-dimethylvaleronitrile)), and LDL (70 μg/ml protein). Astaxanthin dose, dependently significantly prolonged the oxidation lag time (31.5, 45.4, 65.0 min) compared with the control (19.9 min). For the ex vivo study 24 volunteers (mean age 28.2 [SD 7.8] years) consumed astaxanthin at doses of 1.8, 3.6, 14.4 and 21.6 mg per day for 14 days. No other changes were made in the diet. Fasting venous blood samples were taken at days 0, +14. LDL lag time was longer (5.0, 26.2, 42.3 and 30.7% respectively) compared with day O after consuming astaxanthin at doses of 1.8, 3.6, 14.4 and 21.6 mg for 14 days compared with day O, but there was no difference in oxidation of LDL between day O (lag time 59.9 + 7.2 min) and day 14 (57.2 ± 6.0 min) in the control group. Our results provide evidence that consumption of marine animals producing astaxanthin inhibits LDL oxidation and possibly therefore contributes to the prevention of atherosclerosis.

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2, 3-Dihydro-5-hydroxy-2, 2-dipentyl-4, 6-di-tert-butylbenzofuran (BO-653) and probucol, which act as radical scavenging antioxidants, were developed as anti-atherosclerotic medicines. In order to investigate the effect of these antioxidants on cell functions, we analyzed their ability to regulate gene expression in cultured human umbilical vein endothelial cells (HUVECs) using an oligonucleotide chip. Among 6, 416 genes, 17 genes including those encoding mitochondrial proteins and proteins related to oxidative stress response were induced more than 3 fold by BO-653, probucol and tert-butylated hydroquinone (BHQ). On the other hand, genes of three subunits of proteasome (PSMA2, PSMA3, PSMA4) were down-regulated by these antioxidants. A gene of cytochrome P-450 1A1 isozyme, a drug-metabolizing phase I enzyme, was expressed only by BHQ treatment. These results suggested that anti-atherogenic antioxidants affected gene expression in HUVECs by which they might regulate cell functions against oxidative stress.

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This cross sectional study was undertaken to determine whether serum leptin levels were associated with multiple risk factor (MRF) clustering syndrome. We examined the relationship between serum leptin concentrations and blood pressure (BP), serum lipids levels, calculated insulin resistance (HOMA-ratio) and adiposity among 581 Japanese adult women. The serum leptin was increased in female subjects with systolic (≥160 mmHg) and diastolic (≥90 mmHg) hypertension compared with the normotensive females (mean±SE ; 9.3±0.5 vs 7.7±0.3 ; 10.2±0.6 vs 7.1±0.3 ng/ml, both p <0.001). Serum leptin was elevated in those with hyper-cholesterolemia (C ; 220 mg/dl) and triglyceridemia (TG ;≥150 mg/dl) compared with the normolipidemia (9.4±0.4 vs 7.8±0.3 ; 11.7±0.6 vs 7.5±0.2 ng/ml, both p<0.001). Serum leptin was also elevated in those with adiposity (BMI≥ 26.4 kg/m²) and insulin resistance (HOMA-ratio≥2.5) compared with the normal females (14.8±0.7 vs 5.2±0.2 ; 11.3±1.1 vs 7.1±0.4 ng/ml, both p < 0.001). Even after adjusting for BMI or percent body fat mass (BFM), leptin levels remained to be elevated significantly in all these diseases. There was a positive correlation between serum leptin and systolic, diastolic BP, TC, TG, BMI, BFM, IRI and HOMA-ratio (r=0.12, p=0.005 ; r=0.24, p<0.0001 ; r=0.19, p<0.0001; r=0.35, p<0.0001; r=0.72, p<0.0001; r=0.73, p<0.0001; r=0.47, p< 0.0001 ; r=0.44, p<0.0001), and a negative correlation with HDL-C levels (r=-0.20, p< 0.0001). These correlations were also observed in leptin levels after adjusting for the BMI or BFM. Multiple regression analysis showed that BFM, HOMA-ratio and TG were significant determinants of leptin concentration before (t=12.6, p< 0.0001 ; t= 3.33, p= 0.001 ; t= 3.22, p= 0.001) and after adjusting for BMI or BFM.These results suggest that because serum leptin levels were elevated in components of MRF clustering syndrome, leptin may have a pathophysiological role in MRF clustering syndrome.

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Reactive oxygen species (ROS) including superoxide anions (O₂-) play a key role in atherogenesis, and endothelial cells have the ability to generate ROS. To investigate the enzymatic sources of ROS and the effects of lysophosphatidylcholine (LPC), an atherogenic lipid, we measured ROS production in cultured bovine aortic endothelial cells (BAECs) by the lucigenin-enhanced chemiluminescence (CL) method and electron spin resonance (ESR). BAEC homogenates had the enzymatic activity of NADH/NADPH oxidase. BAECs cultured on microcarrier beads generated O₂- under basal conditions. The inhibition of NADH/ NADPH oxidase by diphenylene iodonium (DPI) significantly attenuated O₂- production, whereas no inhibitors of other oxidases suppressed it. Although LPC enhanced O₂ production approximately 3.1-fold, its action was suppressed by DPI. Tyrosine kinase inhibitors significantly attenuated LPC-induced O₂- production. ESR with DMPO demonstrated that LPC increased the formation of the DMPO-hydroxyl adduct in dose- and time-dependent manners. These data suggest that the basal production of O₂- in endothelial cells is mainly mediated by the NADH/NADPH oxidase system and that LPC activates this oxidase to enhance O₂- production through a tyrosine kinase-dependent pathway. The enhancement of ROS production by LPC is probably involved in its atherogenic property.

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