Aim: Type 2 diabetes increases the risk for cardiovascular disease, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) reduce cardiovascular events in these patients. The benefits of statin therapy cannot be explained only by the lipid-lowering effect. The aim of this study was to test the effect of atorvastatin therapy on CD36 scavenger receptor expression, nuclear factor-kappaB (NFκB) levels and markers of inflammation (C-reactive protein, CRP, Tumor Necrosis Factor-α, TNF-α) in circulating monocytes from diabetic patients.
Methods: Twenty-two type 2 diabetic patients were treated for 8 weeks with atorvastatin (20 mg/day). At baseline and after treatment a blood sample was collected for measurement of glucose, lipid profile (total cholesterol, HDL, LDL cholesterol, triglycerides), glycated hemoglobin (HbA1c), CRP and for isolation of monocytes.
Results: Atorvastatin decreased total (p<0.0001) and LDL (p<0.01), and incresased HDL choles-terol (p<0.02). CD36 surface protein expression (anti-CD36 fluorescein isothiocyanate-FITC) was reduced in circulating monocytes after atorvastatin therapy (p<0.02) while immunoblot analysis showed reduced nuclear and increased cytoplasm NFκB levels (p<0.05). Finally, TNFα production in lipopolysaccharide-activated monocytes from patients treated with atorvastatin was reduced (p<0.05).
Conclusion: These results suggest that atorvastatin therapy, beside lowering serum cholesterol levels, could exert anti-atherogenic and anti-inflammatory effects in type 2 diabetic patients.

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Aim: To investigate the factors that influence visceral fat accumulation in adolescence, we performed a medical examination of high school students and assessed abdominal fat thickness and fatty change of the liver.
Methods: A cohort of 374 Japanese high school students aged 15-16 years (193 boys and 181 girls) in public high schools in Chiba prefecture were enrolled. Anthropometric parameters, blood cell count, blood chemistry and adipocytokine levels were measured. Preperitoneal fat thickness (PFT) and echoic contrast of the liver were measured by ultrasonography.
Results: Anthropometric parameters, systolic blood pressure, blood cell count, ALT, AST, FBS, γ-GTP, HDL-C, LpL, UA, adiponectin, resistin and leptin levels differed between sexes. Multivariate regression analysis revealed that leptin was the most appropriate marker for PFT in both sexes (p<0.0001). Visceral obesity, categorized as PFT exceeding 8 mm, was observed in 9.6% of all students. Boys with visceral obesity showed apparent liver dysfunction, hyperlipidemia, hyperinsulinemia, and high leptin and low adiponectin levels. Overall, 16.6% of boys and 30.4% of girls showed hepatorenal echo contrast positivity. Boys with visceral obesity and fatty liver had more risk factors for atherosclerosis.
Conclusions: Physical examination of high school students is important for early detection of atherosclerosis.

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Aim: We investigated how dietary management affected body weight (BW) reduction and energy expenditure in obese and normal-weight type 2 diabetic patients.
Methods: Type 2 diabetic patients who were hospitalized for diabetic control (93 men and 51 women) were checked for resting energy expenditure (REE). Subjects were divided into the two groups according to body mass index (BMI): obese (BMI≥25), and normal-weight (BMI <25). Following the recommendations by JDS, JAS and JASSO, ideal body weight was calculated as [IBW=height (m)×height (m)×22 (kg/m²)], and dietary calorie (kcal/day) was determined as 25 kcal/kg IBW.
Results: Dietary calorie intake during hospitalization was similar in both groups. REE was greater in obese than in normal-weight patients. The difference between the calorie intake and energy expenditure (Δcalorie) was -222±26 kcal in obese patients and 69±27 kcal in normal-weight patients. Obese patients therefore had larger BW decreases than normal-weight patients (-171±12 vs. -92±11 g/day, p<0.005). In the obese group, a positive correlation was found between the change of BW and Δcalorie. This correlation remained after adjusting for age, BMI, gender, and respiratory quotient. Serum lipid profiles were significantly improved in both groups.
Conclusion: These diet instructions showed the appropriate calorie restriction depending on the BMI and induced reasonable BW reduction in both obese and normal-weight subjects. The dietary program recommended by JDS, JAS and JASSO is practically useful for BW control and for improving lipid metabolism in type 2 diabetic patients.

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Aim: HDL has anti-inflammatory effects on macrophages, although the mechanism of action remains unclear. We hypothesized that HDL suppresses the conversion of macrophage-secreted factors into proinflammatory factors via binding, and tried to identify the factor that could form a complex with HDL and/or apolipoprotein (apo) A-I.
Methods and Results: In conditioned media obtained from human monocyte-derived macrophages, we found an apo A-I binding protein and identified the protein as progranulin/proepithelin/acrogranin/PCDGF. Co-immunoprecipitation analysis showing that progranulin binds and forms a complex with apo A-I and the presence of progranulin in the HDL fraction in the sera indicated that progranilin is a novel apolipoprotein. Conditioned media of HEK293 cells transfected with progranulin augmented the expression of TNF-alpha and IL-1-beta on macrophages, but these effects of progranulin were inhibited by co-incubation with HDL or apo A-I. Anti-progranulin antibodies also reduced the expression of TNF-alpha and IL-1-beta on macrophages. Granulins as conversion products derived from progranilin increased TNF-alpha and IL-1-beta expression and the effects were not suppressed by HDL.
Conclusions: Our results suggest that the anti-inflammatory effects of HDL on macrophages might be due to suppression of the conversion of progranulin into proinflammatory granulins by forming a complex.

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Aim: Advanced glycation end products (AGE) play a key role in diabetic vascular complications, whereas matrix metalloproteinases (MMPs) and apoptosis contribute to plaque instability. This study was conducted to investigate the association of AGE-mediated MMP-9 and apoptosis with the renin?angiotensin system (RAS). We also examined the correlation between plasma HbA1c levels and plaque parameters.
Methods: We used autopsy specimens from the aortae and coronary arteries of patients with or without diabetes (n=11, each group) for the immunohistochemistry of AGE, MMP-9, angiotensin-converting enzyme (ACE), and the receptor for AGE (RAGE). Apoptosis was determined by TUNEL staining.
Results: The proportions of AGE accumulation, MMP-9 expression and apoptosis in intimal areas of both aortic and coronary specimens of diabetics were greater than in nondiabetics. MMP-9 expression and apoptosis were correlated with AGE accumulation. RAGE expression was significantly increased in diabetic specimens compared to nondiabetes. Interestingly, the expression of ACE in diabetic specimens was increased and also correlated with AGE accumulation, RAGE expression, MMP-9 expression, and apoptosis in all specimens from diabetics and nondiabetics. Plasma levels of HbA1c were linearly correlated with AGE accumulation, MMP-9, apoptosis, and ACE expression.
Conclusion: The present study shows that AGE/RAGE-related MMP-9 expression and apoptosis were correlated with ACE expression in diabetic plaques and that RAS may be involved in AGE-dependent diabetic vascular complications.

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Aim: Advanced glycation end products (AGE) and a receptor for AGE (RAGE) play a key role in diabetic vascular complications. Matrix metalloproteinases (MMPs) and apoptosis contribute to plaque instability. The renin-angiotensin system (RAS) is crucial for NADPH oxidase-dependent redox signaling pathways in the vascular wall. We investigated the effects of RAS blockade on AGE-triggered signaling pathways and its downstream events, including MMP-9 and apoptosis.
Methods: We used cultured rabbit aortic smooth muscle cells (SMCs), which were stimulated with AGE in the presence or absence of temocaprilat or olmesartan.
Results: Angiotensin converting enzyme (ACE) mRNA levels were increased 4 to 6 hours after adding AGE. AGE induced Rac1 and p47^phox membrane translocation, reactive oxygen species (ROS) generation and NF-κB phosphorylation within 15 minutes, and various molecular expressions after 18 hours, which were attenuated by RAS blockade by temocaprilat or olmesartan. AGE-induced RAGE expression, as well as other molecules, including membrane type 1-MMP (MT1-MMP), monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1), was NADPH oxidase signaling-dependent and blunted by temocaprilat and olmesartan. The parameters of plaque instability, including MMP-9 expression and activity, and apoptosis were up-regulated by AGE, which was markedly attenuated by temocaprilat or olmesartan. Using isolated human monocyte culture, AGE-induced ROS generation and molecular expression were also attenuated by RAS blockade.
Conclusion: The present study shows that AGE-triggered NADPH oxidase signaling pathways, including MMP-9 and apoptosis, were attenuated by RAS blockade, which may be an attractive strategy for treating plaque instability in diabetic vascular complications.

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Aim: In addition to the risk of progression to end-stage renal disease (ESRD), chronic kidney disease (CKD) is also known to be associated with an elevated risk of cardiovascular disease (CVD). Statins may improve renal function in CKD patients.
Methods: The database of the LIVALO Effectiveness and Safety (LIVES) Study, a large-scale (n=20,279), long-term (104 weeks), prospective post-marketing surveillance study of hypercholesterolemic patients treated with pitavastatin, was used to evaluate the effects of pitavastatin on the estimated glomerular filtration rate (eGFR).
Results: Of the 19,925 patients enrolled in the aforementioned study, data from 3,119 patients were analyzed to evaluate the effects of pitavastatin treatment for 104 weeks on the eGFR. In this subanalysis, 958 patients with a baseline eGFR of less than 60 mL/min/1.73 m² (30.7%) were analyzed. A significant increase of the eGFR (+5.4 mL/min/1.73 m²) was observed after 104 weeks of pitavastain treatment (p < 0.001; one-sample t-test). In the analysis of the time-course of changes in the eGFR in response to pitavastatin treatment, the eGFR was elevated by 2.4 mL/min/1.73 m² after 12 weeks' treatment, and by 5.6 mL/min/1.73 m² after 104 weeks' treatment (p < 0.001; repeated measures ANOVA). The results of multivariate analysis identified the presence/absence of proteinuria and the amount change of HDL-C as clinical factors associated with increased eGFR during pitavastatin treatment.
Conclusions: Increased eGFR was noted after 104 weeks of treatment with pitavastatin, which suggests a possible effect of the statin on CKD.

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Aim: Metabolic syndrome (MetS) and postprandial hypertriglyceridemia (PHTG) are closely related and both are associated with coronary heart disease. We have demonstrated that CD36 deficiency is prevalent in the genetic background of MetS and is accompanied by PHTG concomitantly with an increase in remnants and a decrease in high density lipoprotein cholesterol. These findings make CD36 knockout mice (CD36KO) an interesting model for evaluating PHTG in MetS. Fenofibrate was reported to reduce fasting and postprandial triglyceride (TG) levels in hypertriglyceridemic subjects with MetS. To define its mechanism, we investigated the effect of fenofibrate on PHTG in CD36KO.
Methods: Wild-type (WT) and CD36KO mice were fed chow diet and fenofibrate for two weeks. TG concentrations and lipoprotein profiles were assessed during fasting and in the postprandial state in plasma; intestinal mucosa and lymph were collected after oral fat loading for both treatment groups.
Results: Fenofibrate treatment markedly suppressed the postprandial TG response in CD36KO along with decreased apoB-48 levels in plasma. HPLC analysis depicted the decrease of TG content in chylomicrons (CM) and CM remnant-sized lipoproteins contributed to this suppression, suggesting that CM and CM remnant production in the intestines might be attenuated by fenofibrate. ApoB-48 and TG levels in intestinal lymph were markedly reduced after treatment. Intestinal mRNA expression of apoB was also reduced in the postprandial state after fenofibrate administration without affecting any other genes related to CM assembly and production.
Conclusion: Fenofibrate reduces PHTG in CD36KO partially through attenuating intestinal CM production.

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Aim: Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis. Platelet adhesiveness and aggregation increases as a result of smoking. Cigarette smoking modifies haemostatic parameters via thrombosis with a consequently higher rate of cardiovascular events, but smoking-induced alterations of platelet membrane fluidity and other changes have not been studied.
Methods: Thirty experimental and control subjects (mean age 35±8) were selected for the study. Experimental subjects had smoked 10±2 cigarettes per day for 7-10 years. The plasma lipid profile, platelet carbonyls, sulfhydryl groups, Na⁺/k⁺-ATPase activity, fluidity using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), total cholesterol and phospholipids as well individual phospholipids were determined.
Results: Increases in the platelet membrane cholesterol phospholipid (C/P) ratio, phosphotidylethanolamine, phosphotidylserine with decreased phosphotidylcholine, Na⁺/k⁺-ATPase activity, fluidity and no significant change in phosphotidylinositol and sphingomylein, as well as increases in plasma total cholesterol, LDL-cholesterol, protein carbonyls with decreased HDL-cholesterol and sulfhydryl groups were observed in cigarette smokers. Platelet membrane total phospholipids were positively correlated with plasma LDL-cholesterol (r=0.568) and VLDL-cholesterol (r=0.614) in cigarette smokers.
Conclusions: Increased plasma LDL-cholesterol, VLDL-cholesterol and total cholesterol might have resulted in the increased C/P ratio and decreased platelet membrane fluidity of cigarette smokers.

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Aim: Adiponectin has insulin-sensitizing, anti-atherogenic, and anti-inflammatory properties, and researchers have recently reported that ω-3 polyunsaturated fatty acid (PUFA) can increase the serum adiponectin concentration, suggesting that dietary factors, such as fish intake, may have an influence on the serum adiponectin concentration. In general, Japanese subjects consume twice as much fish as people in other countries. We hypothesized that incremental change in serum ω-3 PUFA levels by fish intake is an important regulator of serum adiponectin even in Japanese subjects. The aim of this study was to explore the relationship among fish consumption, serum ω-3 PUFA, such as eicosapentaenoic acid (EPA), levels, and serum adiponectin levels.
Method: We recruited 17 healthy Japanese volunteers (seven men and 10 women) for an 8-week fish-diet intervention (ω-3 PUFA 3.0 g/day) without affecting total energy intake, and measured serum adiponectin concentration and fatty acid profiles.
Results: Fish-diet intervention significantly increased the serum adiponectin concentration in women (from 13.5±4.6 to 15.8±5.2 µg/mL, p <0.01) but not in men (from 8.7±2.8 to 8.7±2.5 µg/mL). Serum ω-3 PUFA increased more in female subjects than male subjects after the fish-diet intervention (57.3±86.6 vs 150.9±46.7 µg/mL, p=0.011), suggesting that changes in ω-3 PUFA concentration may explain the different response between sexes.
Conclusion: A fish-based diet intervention increased the serum adiponectin concentration in young, non-obese, healthy Japanese female subjects. The increment in serum ω-3 PUFA may regulate the serum adiponectin concentration.

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Aim: Seasonal variation in serum lipid levels in the Japanese population remains unclear. The aim of this study was to determine whether a variation in lipid levels exists in Japanese workers.
Methods: We investigated 1,331 employees in our institution (1,192 men, 44±10 years; 139 women, 38±11 years) who underwent health checkups in both June (summer) and December (winter), 2008.
Results: Serum levels of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, and triglyceride were significantly higher in winter than in summer (129.1±31.2 mg/dL versus 125.2±30.2 mg/dL, p<0.0001; 65.9±16.8 mg/dL versus 63.5±16.1 mg/dL, p<0.0001; 110.4±67.5 mg/dL versus 107.5±70.4 mg/dL, p<0.05; respectively), although the ratio of LDL to HDL cholesterol was comparable (2.11±0.81 in summer versus 2.12±0.81 in winter). The frequency of study subjects diagnosed with hypercholesterolemia, defined as LDL cholesterol ≥140 mg/dL, was significantly higher in winter than in summer (34.5 % versus 30.9 %, p<0.0001).
Conclusion: In Japanese workers, we demonstrated that there is a seasonal variation in serum lipid levels and the prevalence of hypercholesterolemia. This result indicates that we have to give careful consideration to the season of blood sampling in the clinical diagnosis of and management decisions for hypercholesterolemia.

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Aim: To examine the relation between lifestyle and the number of metabolic syndrome (MetS) diagnostic components in a general population, and to find a means of preventing the development of MetS components.
Methods: We examined baseline data from 3,365 participants (2,714 men and 651 women) aged 19 to 69 years who underwent a physical examination, lifestyle survey, and blood chemical examination. The physical activity of each participant was classified according to the International Physical Activity Questionnaire (IPAQ). We defined four components for MetS in this study as follows: 1) high BP: systolic BP ≥ 130 mmHg or diastolic BP ≥ 85 mmHg, or the use of antihypertensive drugs; 2) dyslipidemia: high-density lipoprotein-cholesterol concentration < 40 mg/dL, triglycerides concentration ≥ 150 mg/dL, or on medication for dyslipidemia; 3) Impaired glucose tolerance: fasting blood sugar level ≥ 110 mg/d, or if less than 8 hours after meals ≥ 140 mg/dL), or on medication for diabetes mellitus; 4) obesity: body mass index ≥ 25 kg/m².
Results: Those who had 0 to 4 MetS diagnostic components accounted for 1,726, 949, 484, 190, and 16 participants, respectively, in the Poisson distribution. Poisson regression analysis revealed that independent factors contributing to the number of MetS diagnostic components were being male (regression coefficient b=0.600, p < 0.01), age (b=0.027, p < 0.01), IPAQ class (b=-0.272, p= 0.03), and alcohol consumption (b=0.020, p=0.01). The contribution of current smoking was not statistically significant (b=-0.067, p=0.76).
Conclusion: Moderate physical activity was inversely associated with the number of MetS diagnostic components, whereas smoking was not associated.

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Aim: Adipocytes express all components of the renin-angiotensin system (RAS), and adipocyte RAS regulates adipocyte differentiation and metabolism. Plasma angiotensin II (AII) is a putative marker of adipocyte RAS production. The aim of this study was to investigate the effect of pioglitazone on plasma AII in type 2 diabetes (T2D).
Methods: Fifty Japanese subjects with T2D were randomly allocated to two groups. One group was administered pioglitazone 30 mg/day (pioglitazone group) and the other group was not given pioglitazone (control group) for 16 weeks. Lipoprotein lipase mass in preheparin serum (LPL mass) was measured as an adipocyte-derived factor and a marker of insulin sensitivity.
Results: In the pioglitazone group, the mean HbA1c decreased (p<0.0001), LPL mass increased (p<0.0001), and plasma AII decreased (p=0.0007), whereas these parameters were unchanged in the control group. The change in plasma AII correlated negatively with the change in LPL mass (r=-0.312) in the pioglitazone group. In the pioglitazone group, the decrease in plasma AII was higher (p=0.0002) and the increase in LPL mass tended to be higher (p=0.0941) in the subgroup with higher baseline plasma AII than that with lower plasma AII.
Conclusions: The present study indicates that pioglitazone decreases plasma AII associated with an increase in LPL mass in T2D. The insulin-sensitizing effect of pioglitazone may be involved in suppressing adipocyte RAS.

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Aim: Stiffness of the central arteries plays an important role in the pathophysiology of cardiovascular disease, and pulse wave velocity (PWV) of the aorta has been used as the standard measure of central arterial stiffness. An automated device for brachial-ankle (ba) PWV is available, although information is limited whether baPWV reflects the stiffness of central or peripheral arteries. We therefore addressed this question in the present study.
Methods: The subjects were 2,806 consecutive participants in our non-invasive vascular laboratory, excluding those with an ankle-brachial index (ABI) lower than 0.95. PWV measurements were simultaneously performed using an automated device for the ba, heart-femoral (hf, aorta), heart-carotid (hc), heart-brachial (hb), and femoral-ankle (fa) segments. Correlational analyses were performed (1) among these PWV values, (2) between PWV and individual risk factors, and (3) between PWV and the Framingham risk score (FRS), a surrogate index for integrated cardiovascular risk.
Results: The correlation of baPWV was the highest with hfPWV (r=0.796) and the lowest with hcPWV (r=0.541). Among the known factors preferentially affecting central arterial stiffness, higher age, diabetes mellitus, and chronic kidney disease (CKD) were also closely associated with increased baPWV. Finally, FRS was more closely correlated with hfPWV (r=0.613) and baPWV (r=0.609) than with hbPWV (r=0.523), hcPWV (r=0.509), and faPWV (r=0.393).
Conclusion: These results indicate that baPWV is an index of arterial stiffness showing similar characteristics to those of aortic PWV.

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