BACKGROUND:Statins decrease cholesteryl ester transfer protein (CETP) levels, which have been positively associated with hepatic lipid content as well as serum low density lipoproteins-cholesterol (LDL-C) levels. However, the relationship between the CETP status and statin-induced reductions in LDL-C levels has not yet been elucidated in detail. We herein examined the influence of the CETP status on the lipid-reducing effects of pitavastatin in hypercholesterolemic patients with type 2 diabetes mellitus as well as the molecular mechanism underlying pitavastatin-induced modifications in CETP levels.METHODS:Fifty-three patients were treated with 2 mg of pitavastatin for 3 months. Serum levels of LDL-C, small dense (sd) LDL-C, and CETP were measured before and after the pitavastatin treatment. The effects of pitavastatin, T0901317, a specific agonist for liver X receptor (LXR) that reflects hepatic cholesterol contents, and LXR silencing on CETP mRNA expression in HepG2 cells were also examined by a real-time PCR assay.RESULTS:The pitavastatin treatment decreased LDL-C, sdLDL-C, and CETP levels by 39, 42, and 23%, respectively. Despite the absence of a significant association between CETP and LDL-C levels at baseline, baseline CETP levels and its percentage change were an independent positive determinant for the changes observed in LDL-C and sdLDL-C levels. The LXR activation with T0901317 (0.5 μM), an in vitro condition analogous to hepatic cholesterol accumulation, increased CETP mRNA levels in HepG2 cells by approximately 220%, while LXR silencing markedly diminished the increased expression of CETP. Pitavastatin (5 μM) decreased basal CETP mRNA levels by 21%, and this was completely reversed by T0901317.CONCLUSION:Baseline CETP levels may predict the lipid-reducing effects of pitavastatin. Pitavastatin-induced CETP reductions may be partially attributed to decreased LXR activity, predictable by the ensuing decline in hepatic cholesterol synthesis.TRIAL REGISTRATION:UMIN Clinical Trials Registry ID UMIN000019020.

Context: Risk factors for cardiovascular complications in Japanese patients with diabetes have not been fully elucidated.Objective: Our objective was to determine incidence of and risk factors for coronary heart disease (CHD) and stroke in Japanese diabetic patients.Design and Settings: We conducted a prospective study at 59 hospitals throughout Japan.Patients: Patients included 940 men and 831 women with type 2 diabetes (mean age, 58.2 yr) without a history of cardiovascular complications who were followed for a median of 7.86 yr.Intervention: This was an observational study.Main Outcome Measures: Incidence of CHD and stroke was evaluated.Results: Incidences of CHD and stroke per 1000 person-years were 9.59 and 7.45, respectively, whereas those of myocardial and brain infarctions were 3.84 and 6.29, respectively. Multivariate Cox analysis revealed that the serum log-transformed triglyceride level was a potent and independent predictor of CHD [hazard ratio (HR) = 1.54; 95% confidence interval (CI) = 1.22-1.94 per 1 SD increase), comparable to low-density lipoprotein (LDL) cholesterol (HR = 1.49; 95% CI = 1.25-1.78 per 1 SD increase). Triglycerides and LDL cholesterol linearly and continuously increased CHD risk, and subjects in the top third for both had markedly high risks of CHD, and their effects were possibly additive. However, serum triglycerides worked independently of blood pressure levels. Systolic blood pressure was the only significant predictor for stroke except for age (HR = 1.31; 95% CI = 1.04-1.65, per 1 SD increase).Conclusions: In Japanese patients with type 2 diabetes, the serum triglyceride level was a leading predictor of CHD, comparable to LDL cholesterol. Because the serum triglyceride level is not a leading predictor of CHD in diabetic subjects in Western countries, ethnic group-specific strategies for prevention of diabetic macroangiopathy may be indicated. (J Clin Endocrinol Metab 96: 3448-3456, 2011)

AIMThe prognostic power of metabolic syndrome (MetS) in patients with diabetes has been studied with inconsistent results depending on the definition of MetS. To clarify the best combination of MetS components to predict future cardiovascular disease (CVD) events, we estimated CVD risk in Japanese patients with type 2 diabetes according to MetS components.METHODSPatients were categorized according to the presence three MetS components in addition to hyperglycemia. hypertension, dyslipidemia and excess waist circumference (WC) (according to either Japanese or Asian cut-off values). Hazard ratios for CVD events were compared in patients with various categories of MetS components.RESULTSAt least two components of MetS were required for a significantly elevated risk for CVD; however, component combinations with significantly increased risk differed depending on gender or the WC cut-off value. Any two among 1) excess WC (men > or =90 cm, women > or =80 cm); 2) hypertension (systolic blood pressure > or =130 mmHg or diastolic blood pressure > or =85 mmHg or use of an antihypertensive agent); and 3) dyslipidemia (triglycerides > or =150 mg/dL or HDL-cholesterol <40 mg/dL or use of drug treatment) could be used to identify significantly higher risk (approximately twice) for CVD regardless of gender.CONCLUSIONSThe results suggest that the current MetS criteria should be modified when applied to patients with type 2 diabetes.

Excess waist circumference (WC) is a frequently used indicator of abdominal obesity and/or cardiovascular disease (CVD) risk. Nonetheless, search of the literature revealed no prospective studies on the association between WC and CVD events in diabetic patients. In this study, the clinical significance and implications of WC as a cardiovascular and metabolic risk indicator was prospectively investigated in Japanese patients with type 2 diabetes. For this purpose, baseline data on WC, hypertension, and dyslipidemia were collected and subsequent CVD (coronary heart disease and stroke) events during the following 8 years were studied in 1,424 Japanese type 2 diabetic patients, and the cross‐sectional/longitudinal associations between WC and CVD risk factors/events were analyzed. Mean WC levels were significantly increased according to the number of coexisting risk factors. However, no significant difference in mean WC between subgroups with and without CVD events was noted, and excess WC alone was not predictive of subsequent CVD events either in male or female subjects even after adjustment for age, smoking, hypertension, and dyslipidemia. In female patients, excess WC (≥80 cm) was predictive of CVD events only with the coexistence of hypertension. In Japanese diabetic patients, excess WC alone, although a good marker for clustering of CVD risk factors, did not raise the risk of CVD events unless accompanied by hypertension in female patients. Further investigations are necessary before WC as a risk factor can be utilized in clinical settings for the management of diabetes in this population.

[Background] Percutaneous coronary intervention (PCI) is useful for the treatment of coronary artery disease (CAD). The clinical outcome of the follow-up PCI revealed that the incidence of CAD (de novo lesion) still remained. This new lesion is considered to be the atheroma caused by the intimal injury incurred during the PCI procedure. We evaluated the therapeutic effect of LDL-apheresis on CAD after PCI retrospectively. [Method] The subjects were 11 familial hyperchoresterolemic patients (FH) who underwent LDL-apheresis (LA-15) monthly for 5 years and whose stenotic lesions (AHA Type B and/or C) matched 32 patients (C) who underwent conventional therapy. All patients underwent the first PCI and, within the following 5 years, all of the segments were evaluated by coronary angiograpy. The atheromatous lesion was recognized as a minimal lesion in diameter and was defined according to the segment criteria by AHA. After a 5-year follow-up, the incidence of cardiac events such as angina, myocardial infarction and cardiac death, the incidence of de novo lesions and the incidence of restenotic lesions following PCI were evaluated between two groups. [Results] The incidence of the cardiac events was significantly lower in FH (27.3% vs 56.2%). In FH, de novo lesions were only 4 segments; however, in C, de novo lesions were 16 segments. [Conclusion] These clinical outcomes suggest that LDL-apheresis therapy may be useful for reducing CAD following PCI.

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation–inflammation–thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IκB kinase β (IKKβ) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-κB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKβ and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKβ and ERK2, activations of NF-κB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKβ/proteasome/NF-κB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation–inflammation–thrombosis circuit during infections by VT-producing E. coli.

Plasminogen activator inhibitor-1 (PAI-1) may contribute to renal fibrosis because of its involvement in matrix (ECM) accumulation through inhibition of plasmin-dependent ECM degradation. The aim of this study is to determine urinary PAI-1 concentrations and its intrarenal localization in patients with various renal diseases and to identify inducers for PAI-1 expression in human cultured proximal renal tubular cells (HRCs). Urinary PAI-1 concentrations were significantly higher in patients with overt diabetic nephropathy (DN, n=36) than in proliferative glomerulonephritis (PGN, n=8), nephrotic syndrome (NS, n=10) and healthy controls (n=12). Urinary PAI-1 concentrations (ng/gCr) were directly correlated with urinary N-acetyl glucosaminidase (NAG) levels (r=0.58, p<0.05). As for intrarenal localization of PAI-1 antigen, strong stainings for PAI-1 were observed in proximal tubular cells of renal biopsy samples from patients with DN, while no stainings for PAI-1 were found in renal tissues of PGN or NS. Immunoblot analysis revealed the presence of PAI-1 protein in whole cell lyzates from HRCs grown to semiconfluency. Exposure of growth-arrested HRCs with hypoxia (1% O2) or TNF-alpha (10 ng/ml) for 24 hours increased the secretion rate of PAI-1 protein by about 2.0-fold, while 24-hour treatment with high glucose (450 mg/dl) did not increase PAI-1 secretion at all, compared with that of the control cells under normal glucose (100 mg/dl) and normoxia (18% O2). These findings suggest that PAI-1 expression is upregulated especially in the proximal renal tubular cells of DN, which may be explained partially by hypoxia and inflammatory cytokines but not high glucose.

PURPOSE To compare the contributions of physical activity volume (vPA) and intensity (iPA) to HDL levels in middle-aged women. METHODS A cross-sectional design was used. Subjects were 273 women, mean age 40.1 years, who were nonsmokers and non-obese (BMI <30). HDL was measured by a certified hospital laboratory using the Dimension clinical chemistry system. vPA and iPA were assessed using MTI (formerly CSA) accelerometers worn over the left hip for 7 consecutive days. Each day was divided into 10-minute segments (epochs) for a total of 144 epochs each day and 1008 epochs over the 7 days. The sum of all activity counts over the 1008 epochs was used to index vPA. Subjects were divided into quartiles based on their vPA and the middle-two quartiles were collapsed producing 3 categories of vPA. iPA was calculated by categorizing the activity counts of every epoch as Low Intensity, if the activity counts for that epoch were <30,000 (sedentary to slow walking), Moderate Intensity, 30,000–50,000 counts (slow to fast walking), or High Intensity, >50,000 counts (fast walking to running and beyond). A total of 9 epochs (90 min of activity) over the week had to be accumulated in the High Intensity category for the woman to be classified into that iPA category. If she did not have 90 min of High Intensity activity, the 9 epoch cut-point was used to check for Moderate Intensity. If there was not 90 min of activity within the High or Moderate Intensity categories, then she was classified into the Low Intensity category. RESULTS Mean (± SD) vPA over the week was 2.7 ± 0.8 million activity counts. A total of 145 (53.1%) women were classified in the Low Intensity category, 70 (25.6%) in the Moderate Intensity category, and 58 (21.2%) in the High Intensity category. With age controlled, HDL levels differed across the three vPA categories F=4.1, p=0.044), but not without (F=3.5, p=0.063). On the other hand, HDL levels differed across the iPA categories with age controlled (F=6.9, p=0.009), and with no adjustment for differences in age (F=6.2, 0.014). Specifically, after controlling for age, mean HDL levels were 56.2, 52.2, and 52.1 across the High, Moderate, and Low Intensity categories, respectively, with the High Intensity subjects showing significantly higher levels of HDL than the other two categories of women. After controlling for age and iPA, the association between vPA and HDL was eliminated (F=1.1, p=0.30). However, after adjusting for age and vPA, the relationship between iPA and HDL remained significant (F=4.0, p=0.047). CONCLUSION When weekly duration of activity is at least 90 minutes, intensity of physical activity seems to contribute more to HDL levels in women than total volume of activity.

To explore the functional effects of hormone-sensitive lipase (HSL) in diacylglycerol (DAG) metabolism, Chinese hamster ovary cells were stably transfected with rat HSL cDNA (wt-HSL), inactive mutant S423A-HSL cDNA (S423A) and pcDNA3 vector alone (Ct). [(14)C]Glucose-incorporation into triglyceride (TG) was 75% lower in the presence or absence of insulin in cells expressing wt-HSL compared to Ct or S423A. [(14)C]Glucose-incorporation into DAG was 33% lower without insulin and 51% lower with insulin in cells expressing wt-HSL compared to Ct or S423A. Insulin stimulated glucose-incorporation into DAG 2.2-fold in S423A and Ct cells, whereas only a 50% increase was observed in cells expressing wt-HSL. Phospholipase C-mediated release of DAG from membrane phospholipids was reduced 70% in cells expressing wt-HSL compared to Ct or S423A. Western blot analysis showed that membrane-bound protein kinase C (PKC)-alpha and -epsilon were decreased 40-50% in cells expressing wt-HSL grown in high glucose with insulin. These data show that HSL potentially hydrolyzes cellular DAG generated either by de novo synthesis from glucose or release from membrane phospholipids by phospholipase C, resulting in a reduction in the translocation of DAG-sensitive PKCs.

Biological membranes organize and compartmentalize cell signaling into discrete microdomains, a process that often involves stable, cholesterol-rich platforms that facilitate protein–protein interactions. Polarized cells with distinct apical and basolateral cell processes rely on such compartmentalization to maintain proper function. In the cochlea, a variety of highly polarized sensory and non-sensory cells are responsible for the early stages of sound processing in the ear, yet little is known about the mechanisms that traffic and organize signaling complexes within these cells. We sought to determine the prevalence, localization, and protein composition of cholesterol-rich lipid microdomains in the cochlea. Lipid raft components, including the scaffolding protein caveolin and the ganglioside GM1, were found in sensory, neural, and glial cells. Mass spectrometry of detergent-resistant membrane (DRM) fractions revealed over 600 putative raft proteins associated with subcellular localization, trafficking, and metabolism. Among the DRM constituents were several proteins involved in human forms of deafness including those involved in ion homeostasis, such as the potassium channel KCNQ1, the co-transporter SLC12A2, and gap junction proteins GJA1 and GJB6. The presence of caveolin in the cochlea and the abundance of proteins in cholesterol-rich DRM suggest that lipid microdomains play a significant role in cochlear physiology.Although mechanisms underlying cholesterol synthesis, homeostasis, and compartmentalization in the ear are poorly understood, there are several lines of evidence indicating that cholesterol is a key modulator of cochlear function. Depletion of cholesterol in mature sensory cells alters calcium signaling, changes excitability during development, and affects the biomechanical processes in outer hair cells that are responsible for hearing acuity. More recently, we have established that the cholesterol-modulator beta-cyclodextrin is capable of inducing significant and permanent hearing loss when delivered subcutaneously at high doses. We hypothesize that proteins involved in cochlear homeostasis and otopathology are partitioned into cholesterol-rich domains. The results of a large-scale proteomic analysis point to metabolic processes, scaffolding/trafficking, and ion homeostasis as particularly associated with cholesterol microdomains. These data offer insight into the proteins and protein families that may underlie cholesterol-mediated effects in sensory cell excitability and cyclodextrin ototoxicity.

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Although peroxisome proliferator-activated receptor alpha (PPARalpha) is closely associated with myocardial fatty acid metabolism, the pathophysiological role of PPARalpha in myocardial infarction (MI) is not yet known. The aim of the present study was to clarify the relationship between cardiac energy metabolism and PPARalpha expression in the remodelling of myocardium after MI. We assayed the expression of PPARalpha and several metabolic genes in cultured cardiac cells (myocytes and nonmyocytes) and in MI hearts. PPARalpha was strongly expressed in cardiac myocytes but not in nonmyocytes (mainly fibroblasts). In MI rats, PPARalpha and PPARalpha-regulated genes (lipoprotein lipase, heart-type fatty acid binding protein, long-chain acyl-CoA dehydrogenase and uncoupling protein-3) were decreased concomitantly, whereas uncoupling protein-2 was not decreased in severely ischemic regions. Immunohistochemical staining for PPARalpha was less decreased in borderline myocardium than in sham-operated hearts. Furthermore, in electron microscopic study, there were no lipid droplet accumulations in surviving myocardium after MI. Our results suggest that the reduced expression of PPARalpha is closely related to that of fatty acid metabolism genes in infarcted myocardium, and PPARalpha may play an important role in cardiac energy metabolism during remodelling after MI.