Aim: Adherence to a Mediterranean diet has been shown to lower the risk of developing several chronic diseases. The ability to augment circulating adiponectin levels is proposed as an underlying mechanism mediating the beneficial effects of this diet. We aimed to examine whether the positive relationship between the Mediterranean diet and adiponectin is altered by cigarette smoking, taking potential confounders into consideration.
Methods: Plasma adiponectin levels were enzymatically measured in 45 never smokers, 61 smokers and 34 ex-smokers who adhered to a Mediterranean style diet and in 41 never smokers who did not adhere to the diet.
Results: Plasma adiponectin levels increased significantly in nonsmoking diet adherents compared to nonsmoking non-diet adherents. Among the diet adherents adiponectin decreased significantly in both moderate and heavy smokers compared to never smokers and significantly increased in quitters compared to smokers. Multiple regression analysis, controlling for age, obesity, Mediterranean diet and insulin resistance revealed an independent inverse association of smoking with adiponectin. Adiponectin levels remained significant and similar in subjects stratified according to age (</>50 years), BMI (</>25 kg/m²) and HOMA-IR (</>1.6).
Conclusions: Despite its positive effects on adiponectin, the Mediterranean diet failed to negate the adiponectin-lowering effect of cigarette smoking, demonstrating the profound and independent capacity of cigarette smoke to negatively influence human health.

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Aim: Statins have many anti-atherogenic effects apart from reducing the serum level of low density lipoprotein cholesterol (LDL-C). For instance, statins can enhance the expression of endothelial nitric oxide synthase (eNOS), at least partly by upregulating its transcription. Although it has been reported that -786 T/C polymorphism of the promoter region has an important influence on statininduced transcription of the human eNOS gene, much remains unclear about statin-induced eNOS transcription. We tried to identify other statin-responsive promoter regions.
Methods: A human endothelial cell line (EA.hy926 cells) was treated with pitavastatin, atorvastatin, or fluvastatin, after which eNOS mRNA levels were assessed by quantitative real-time RT-PCR. EA.hy926 cells were also transiently transfected with luciferase reporter genes driven by various lengths of the human eNOS promoter and were treated with statins before luciferase activity was measured.
Results: Statin treatment increased eNOS mRNA levels in EA.hy926 cells. In addition, cells transfected with the reporter gene driven by the eNOS promoter fragment starting from position -740 exhibited a pitavastatin-induced increase of luciferase activity, which was not observed in cells transfected with the reporter gene driven by the fragment starting from -727. Similar results were also obtained with atorvastatin and fluvastatin.
Conclusions: Statins enhanced eNOS expression in EA.hy926 cells, at least partly by inducing its transcription. Although a statin-responsive sequence that could function even in a heterologous promoter was not precisely identified, the region of the human eNOS promoter around position -730 seems to be critical for statin-induced transcriptional activation.

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Aim: Individuals with type 2 diabetes are at high risk for cardiovascular events; however, a modality that could reduce risk has not been fully evaluated. We examined the annual incidence rate of cardiovascular events in asymptomatic Japanese diabetic patients who underwent gated myocardial perfusion single-photon computed tomography (SPECT).
Methods: Asymptomatic patients (n= 485) aged ≥50 years and with either a maximal carotid artery intima-media thickness of ≥1.1 mm, or urinary albumin ≥30 mg/g creatinine, or at least two of abdominal obesity, low HDL cholesterol, high triglyceride levels and hypertension were evaluated at 50 institutions using gated SPECT with the stress-rest protocol and were followed up for three years. In the patients with summed stress scores (SSS) of ≥9, the initial high cardiovascular incidence rate in the first year declined significantly (p<0.001) from 21% to 2% during the second and third year.
Results: The incident rate was consistent among diabetics with low SSS during follow-up. Baseline LDL and non-HDL cholesterol levels significantly reduced (123±6.4 to 99±6.1 mg/dL; p<0.05) during the first year in diabetic patients with high SSS, but did not significantly change in diabetic patients with low (<9) SSS.
Conclusion: The immediate improvement of lipid levels within one year correlated with the significant reduction in CVD events arising in diabetic patients with moderate or severe myocardial ischemia. Thus, rapidly lowering hyperlipidemia is very effective for patients with type 2 diabetes at high risk for cardiac events.

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Aim: Visceral fat accumulation plays an integral role in morbidity and mortality rates by increasing the risk of developing metabolic disorders such as type 2 diabetes, dyslipidemia, and hypertension. New genetic loci associated with fat distribution, measured by waist-hip ratios and computed tomography (CT), have recently been identified by genome-wide association studies in European-descent populations. This study used CT to investigate whether single nucleotide polymorphisms (SNPs) that confer susceptibility to fat distribution are associated with visceral fat area (VFA) and subcutaneous fat area (SFA) in the Japanese population.
Methods: We measured the VFAs and SFAs of 1424 obese Japanese subjects (BMI≥25 kg/m², 635 men and 789 women) that were genotyped at 15 SNPs, namely, TBX15 rs984222, DNM3 rs1011731, LYPLAL1 rs4846567, GRB14 rs10195252, NISCH rs6784615, ADAMTS9 rs6795735, CPEB4 rs6861681, LY86 rs1294421, VEGFA rs6905288, RSPO3 rs9491696, NFE2L3 rs1055144, ITPR2 rs718314, HOXC13 rs1443512, ZNRF3 rs4823006 and THNSL2 rs1659258.
Results: The G-allele of LYPLAL1 rs4846567 was borderline associated with an increased ratio of VFA to SFA (V/S ratio; p= 0.0020). LYPLAL1 rs4846567 had a stronger effect on the V/S ratio in women (p= 0.0078) than in men (p= 0.12); however, neither result was significant after Bonferroni correction for multiple comparisons. NISCH rs6784615 was nominally associated with increased VFA (p=0.040) and V/S ratio (p= 0.020). The other SNPs analyzed were not significantly associated with body mass index (BMI), VFA, or SFA.
Conclusion: Our results suggest that LYPLAL1 rs4846567 and NISCH rs6784615 may influence fat distribution in the Japanese population.

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Aim: Using general Japanese populations, we performed a replication study of genetic loci previously identified in European-descent populations as being associated with uric acid and gout. The relative contribution of non-genetic and genetic factors to the variances in serum uric acid concentration was then evaluated.
Methods: Seven single nucleotide polymorphisms (SNPs) were genotyped from 7 candidate loci robustly confirmed in Europeans. Genotyping was performed in up to 17,226 individuals, from which 237 hyperuricemia cases and 3,218 controls were chosen for a case-control study. For 6 SNPs showing a replication of uric acid association in 17,076 general population samples, we further tested the associations with other metabolic traits (n≤5,745) and with type 2 diabetes (931 cases and 1404 controls) and coronary artery disease (806 cases and 1337 controls).
Results: Significant uric acid associations (one-tailed p<0.05) were replicated for 6 loci in Japanese. The strongest association was detected at SLC22A12 rs505802 for uric acid (p=2.4×10⁻⁵⁰) and ABCG2 rs2231142 for hyperuricemia (p3.6×10⁻¹⁰). The combined genetic effect could explain some proportion of inter-individual variation in uric acid (R²=0.03) and was more or less comparable to the effect of well-recognized risk factors −BMI (R²=0.04) and alcohol intake (R²=0.01). The tested SNPs were not significantly associated with cardiovascular risk traits except for GCKR rs780094.
Conclusion: Our results confirm that 6 common uric acid variant loci are reproducible in Japanese. Further investigation is warranted to efficiently use the knowledge about genetic factors in combination with modifiable risk factors when we decide an individual’s treatment strategy for hyperuricemia.

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Aims: While statins have the property of increasing high-density lipoprotein cholesterol (HDL-C) in addition to lowering low-density lipoprotein cholesterol (LDL-C), a potential adverse effect on glucose metabolism has raised a concern over statin therapy. In a comparative trial, we investigated the effects of low-dose pravastatin and atorvastatin on HDL-C and glucose metabolism in patients with elevated LDL-C levels and glucose intolerance.
Methods: Eligible patients were men aged ≥20 years or postmenopausal women who had LDL-C ≥140 mg/dL, HDL-C <80 mg/dL, and triglycerides <500 mg/dL and who had glucose intolerance. The patients were randomly allocated to either pravastatin (10 mg/day) or atorvastatin (10 mg/day) treatment for 12 months in an unblinded fashion. The percent changes from the baseline were compared between the treatments.
Results: Of 202 patients who were randomized to either of the two treatments, 195 patients started the study medication, and 187 patients underwent the follow-up measurements at 6 or 12 months (pravastatin, n= 93; atorvastatin, n= 94). HDL-C increased by 4.3% (p= 0.03) in the pravastatin group and by 5.8% (p=0.0005) in the atorvastatin group and showed no between-group difference (p= 0.38). LDL-C decreased substantially in both groups (pravastatin, 21.5%; atorvastatin, 35.5%), and the decrease was much greater in the atorvastain group (p<0.0001). HbA1c slightly increased in both groups, but showed no measurable difference in the increase between the two treatments (p=0.30).
Conclusion: Pravastatin and atorvastatin of 10 mg per day each increased HDL-C by almost the same extent. These two statins did not show a differential effect on glucose metabolism.

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Aim: Apolipoprotein F (apo F), also known as lipid transfer inhibitory protein (LTIP), is a protein component of plasma lipoprotein classes including HDL and functions to inhibit lipid transfer between lipoproteins in vitro. To study the role of plasma apo F, a reliable and sensitive tool for the quantification would be needed.
Methods: We have developed a sandwich ELISA using two monoclonal antibodies for human plasma apo F, and analyzed apo F concentration in 397 Japanese healthy and 221 hypertriglyceridemic subjects.
Results: Our ELISA enables apo F to be assayed in the range of 0.6-25 µg/mL with intra- and inter-assay coefficients of variation less than 3.8% and 7.8%, respectively. In healthy subjects, plasma apo F concentration was 12.5±2.9 µg/mL (mean±SD), and was significantly higher in females than in males (p<0.05). By linear regression analysis in healthy subjects, plasma apo F concentration correlated positively with HDL cholesterol and apo A-I levels, and in males but not in females, negatively with apo B and triglyceride levels. It also correlated negatively with intrinsic CETP activity measured using intrinsic apo B-containing lipoprotein as an acceptor, and positively with PLTP mass and apo J levels. Apo F concentration in hypertriglyceridemic patients (10.3±3.1 µg/mL) was lower than in healthy controls (p<0.0001) and correlated positively with PLTP mass.
Conclusions: Our ELISA is reliable and sensitive for the quantification of plasma apo F concentration. This system can be applicable for clinical significance in lipoprotein metabolism and reverse cholesterol transport.

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Aim: Arterial stiffness has been reported to correlate with cardiovascular disease (CVD). Brachial-ankle pulse wave velocity (baPWV) is easy to measure and has been used as a marker to evaluate arterial stiffness. The objective of the present study was to determine the cut-off value of baPWV for predicting cardiovascular prognosis in a prospective cohort study.
Methods: Four hundred forty patients with essential hypertension were analyzed in study 1 with a mean follow-up of 6.3±0.1 years. Four hundred patients from study 1 who did not have a past history of CVD and/or stroke were analyzed in study 2 with a mean follow-up of 6.4±0.1 years. Stroke, CVD, and death were the primary endpoints.
Results: Receiver operating characteristic (ROC) curve analysis revealed that 1750.0 cm/sec is an appropriate cut-off value for baPWV to predict the onset of stroke, CVD, stroke+CVD, and total mortality (area under curve: 0.576-0.719). A baPWV higher than 1750.0 may also be a significant and independent risk factor for the onset of CVD+stroke (relative risk: 2.048 (1.176-3.616), p= 0.0113 in study 1; relative risk: 1.920 (1.028-3.634), p=0.0408 in study 2).
Conclusions: The present study indicates that 1750.0 cm/sec could be a useful cut-off value for baPWV to predict cardiovascular prognosis.

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