Yuji Hirowatari

Release reaction of brain-derived neurotrophic factor (BDNF) through PAR1 activation and its two distinct pools in human platelets

Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets. Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting. BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 μM PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in α-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to α-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation. In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the α-granules and cytoplasm of human platelets, and only BDNF in α-granules is released through platelet activation.

Serotonin levels in platelet-poor plasma and whole blood in people with type 2 diabetes with chronic kidney disease

AIMS Patients with diabetes mellitus (DM) are prone to atherosclerosis. Atherosclerosis activates platelets; activated platelets release serotonin, and therefore, evaluation of serotonin levels in blood could be a valuable biomarker for future risk of cardiovascular events. METHODS Plasma serotonin levels obtained from patients with DM complicated with chronic kidney disease were measured using HPLC and were compared to serotonin levels of healthy control subjects. Patients with DM were classified into 2 subgroups of mildly (group 1) and moderately/severely (group 2) impaired renal function. RESULTS Serotonin concentration in platelet-poor plasma for group 1 was significantly higher than that of healthy control subjects (p < 0.01), and was significantly higher than that of patients from group 2 (p < 0.05). The concentration of serotonin in whole blood for group 2 patients was significantly lower than that measured from healthy control subjects (p < 0.01). The ratio of the plasma to whole blood level was significantly elevated in both groups 1 and 2 compared with healthy controls (p < 0.01). CONCLUSIONS Our results indicate that platelets are activated to release serotonin into plasma in diabetic patients with mildly impaired renal function. When renal damage is advanced, platelets are over-activated to release serotonin.

Effects of Diacylglycerol on Glucose, Lipid Metabolism, and Plasma Serotonin Levels in Lean Japanese

Objective: Diacylglycerol (DAG)‐rich oil has been suggested to suppress postprandial hyperlipidemia and promote negative caloric balance by increasing energy expenditure (EE), due to small intestine physiochemical dynamics that differ from triacylglycerol (TAG). We studied the effect of DAG on postprandial glucose/insulin metabolism by loading of carbohydrate with oil. Further, to reveal the mechanism for increased EE by DAG, we measured plasma serotonin, which is mostly present in the small intestine and mediates peripheral sympathetic thermogenesis.

Serotonin in peripheral blood reflects oxidative stress and plays a crucial role in atherosclerosis: Novel insights toward holistic anti-atherothrombotic strategy.

We enrolled 132 outpatients with cardiovascular risk factors to evaluate the serotonin levels in platelet-poor plasma (PPP) and whole blood (WB). PPP serotonin levels and PPP/WB serotonin ratio were significantly correlated with levels of oxidative stress measured by derivative reactive oxygen metabolites (d-ROM). Twenty-five subjects were revealed to have stable coronary artery disease (CAD), and the levels CRP, d-ROM, and PPP/WB serotonin ratio were significantly higher in subjects with CAD than in those without CAD. Logistic regression analysis performed with the endpoint of having CAD revealed that the PPP/WB serotonin ratio was independently associated with CAD (odds ratio 3.37, 95% confidence interval 1.04-10.9, P = 0.04). Receiver operating characteristic (ROC) curve analyses to discriminate subjects with CAD from those without CAD indicated that combining PPP/WB serotonin ratio and d-ROM improved diagnostic utility. Targeting the serotonin-oxidative stress axis as part of a holistic anti-atherothrombotic strategy could be beneficial for patients with atherosclerosis.

Analytical evaluation of plasma serotonin and sphingosine 1-phosphate and their clinical assessment in early atherosclerosis.

ObjectivesSerotonin stored in platelets is released into plasma on aggregation and activation in atherosclerotic diseases. Sphingosine 1-phosphate (S1P) in plasma is mainly derived from red blood cells and is responsible for the production of nitric oxide in endothelial cells and protects vasculature. The purpose of this study was to investigate the plasma levels of serotonin, S1P, and their clinical relationships with vascular endothelial function in patients with early atherosclerosis. MethodsBlood was withdrawn from patients with low-to-moderate risks of atherosclerotic diseases (n=49, 39±7 years). Platelet-poor plasma was immediately centrifuged. Serotonin levels in plasma were measured with high-performance liquid chromatography. S1P levels in plasma were measured by high-performance liquid chromatography after fluorescent derivatization with o-phthaldialdehyde. Endothelial function was assessed by endothelium-dependent flow-mediated dilation (FMD) and endothelium-independent dilation was measured by glycerol trinitrate-induced dilation using an ultrasound system. ResultsPlasma serotonin was inversely correlated with the FMD value (r=−0.287, P<0.05). Fourteen patients with dyslipidemia, who had not shown improvements after lifestyle modifications, were subsequently treated with rosuvastatin (2.5 mg/day). After 4 weeks of treatment, rosuvastatin improved lipid profiles. Rosuvastatin increased FMD, whereas glycerol trinitrate-induced dilation was unchanged. Notably, percentage decrease in plasma serotonin was inversely correlated with percentage increase in plasma S1P (r=−0.557, P<0.05). ConclusionPlasma serotonin was inversely correlated with FMD and a decrease in plasma serotonin was inversely correlated with an increase in plasma S1P after statin treatment. The results suggested that plasma levels of serotonin and S1P may be useful for the assessment of endothelial function of patients with low-to-moderate risks of atherosclerotic diseases.

Characteristic comparison of triglyceride-rich remnant lipoprotein measurement between a new homogenous assay (RemL-C) and a conventional immunoseparation method (RLP-C)

BackgroundIncreased serum remnant lipoproteins are supposed to predict cardiovascular disease in addition to increased LDL. A new homogenous assay for remnant lipoprotein-cholesterol (RemL-C) has been developed as an alternative to remnant-like particle-cholesterol (RLP-C), an immunoseparation assay, widely used for the measurement of remnant lipoprotein cholesterol.MethodsWe evaluated the correlations and data validation between the 2 assays in 83 subjects (49 men and 34 women) without diabetes, hypertension and medications for hyperlipidemia, diabetes, and hypertension, and investigated the characteristics of remnant lipoproteins obtained by the two methods (RLP-C and RemL-C) and their relationships with IDL-cholesterol determined by our developed HPLC method.ResultsA positive correlation was significantly found between the two methods (r = 0.853, 95%CI 0.781–0.903, p < 0.0001). Bland & Altman analysis revealed that RemL-C values were likely to be significantly higher than RLP-C values, particularly in samples with high levels of remnant lipoproteins. Several data dissociations between the RemL-C and RLP-C were also observed. The HPLC chromatograms show high concentrations of chylomicron cholesterol in serum samples with RemL-C level < RLP-C level, but high concentrations of IDL-cholesterol in samples with RemL-C level > RLP-C level. RemL-C (r = 0.339, 95%CI 0.152–0.903; p = 0.0005) significantly correlated with IDL-cholesterol, but not RLP-C (r = 0.17, 95%CI -0.047–0.372; p = 0.1237) in all the samples (n = 83).ConclusionThese results suggest that there is generally a significant correlation between RemL-C and RLP-C. However, RemL-C assay is likely to reflect IDL more closely than RLP-C.

Implications of decreased serum adiponectin for type IIb hyperlipidaemia and increased cholesterol levels of very-low-density lipoprotein in type II diabetic patients.

The present study was performed to investigate the relevance of cholesterol levels of plasma lipoproteins [HDL (high-density lipoprotein), LDL (low-density lipoprotein), IDL (immediate-density lipoprotein), VLDL (very-LDL) and chylomicrons] determined by a novel HPLC method, with adiponectin, which is decreased in Type II diabetes and assumed to be involved in dysregulated metabolism and atherogenesis. Type II diabetic patients who were not treated with insulin, statins and fibrates were enrolled. Study subjects included Type II diabetic patients with normolipidaemia (DM-NL; n=15), type 4 hyperlipidaemia (DM-T4HL; n=13), Type IIa hyperlipidaemia (DM-T2aHL; n=15) and Type IIb hyperlipidaemia (DM-T2bHL; n=13). Fasting blood samples were collected. The serum adiponectin level was lower in DM-T2bHL than in any of the other groups. Cholesterol levels of each lipoprotein fraction, serum triacylglycerol (triglyceride), remnant-like particle-cholesterol, fasting plasma glucose, HbA1c (glycated haemoglobin), age, gender difference and BMI (body mass index) were incorporated into a stepwise regression analysis as independent variables. VLDL-cholesterol correlated inversely with adiponectin independently of age, BMI, gender difference and glycaemic control. Although the mechanisms remain to be explored, serum adiponectin was reduced particularly in Type II diabetics with type IIb hyperlipidaemia and correlated inversely with VLDL-cholesterol. Measuring VLDL-cholesterol may be helpful for understanding the pathological features of diabetic dyslipidaemia.

Relevance of intermediate-density lipoprotein cholesterol to Framingham risk score of coronary heart disease in middle-aged men with increased non-HDL cholesterol

BACKGROUND Cholesterol levels of non-high-density lipoprotein (non-HDL), which contains low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL) and chylomicron (CM) remnant, have been proven to perform a significant predictor of coronary heart disease (CHD) better than LDL-cholesterol regardless of triglyceride (TG) levels. The present study investigated the relevance of TG-rich lipoproteins (IDL, VLDL, CM) to Framingham risk score (FRS) predictive of 10-year CHD risk. METHODS Lipoprotein profiles (cholesterol levels of HDL, LDL, IDL, VLDL, CM) in Japanese men (n = 487) who underwent medical check-up were determined by using our developed anion-exchange high performance liquid chromatography (AEX-HPLC). Total-cholesterol (TC), TG, fasting blood sugar (FBS) and hemoglobin (Hb) A1c were measured by routine methods. The lipoprotein profiles, non-HDL-cholesterol, TC, and TG were examined on these associations with FRS. RESULTS The lipid levels except for CM-cholesterol were significantly different between two groups (low FRS, < 10%; high FRS, ≥10%) (P < 0.0001), and body mass index (BMI), TC, TG, IDL-, and VLDL-cholesterol were significantly and positively correlated with FRS. Among them, the significant association of non-HDL-cholesterol to FRS was noted (r = 0.411, P < 0.0001). Multiple stepwise regression analysis shows that, in addition to non-HDL-cholesterol, IDL-cholesterol in TG-rich lipoproteins was significantly correlated with FRS in independently of BMI. These correlation results were similarly found even when the part of the study subjects (n = 348) without the drug therapy for hyperlipidemia, diabetes, and hypertension was investigated. CONCLUSIONS These results suggest that IDL-cholesterol may serve as a useful marker for CHD risk in Japanese men with increased non-HDL-cholesterol.

Analysis of cholesterol levels in lipoprotein(a) with anion-exchange chromatography

We previously established an analysis method for determining the cholesterol levels of five major lipoprotein classes [HDL, LDL, intermediate density lipoprotein (IDL), VLDL, and chylomicrons] in serum by an anion-exchange (AEX)-HPLC method, but lipoprotein(a) [Lp(a)], a well-known risk factor for atherosclerotic diseases, was not determinable. Therefore, we established new AEX-HPLC separation conditions for analyzing the cholesterol levels of six lipoprotein classes, including Lp(a). Serum lipoproteins were separated by HPLC with a diethylaminoethyl-ligand nonporous polymer-based column by elution with a stepwise gradient of the sodium perchlorate concentration. In this improved method, HDL, LDL, IDL, VLDL, chylomicrons, and Lp(a) were each eluted from the column. The cholesterol levels of the eluted lipoproteins were measured enzymatically by a postcolumn reaction. The within-day assay and between-day assay coefficients of variation for the lipoprotein cholesterol levels were in the ranges of 0.29–11.86% and 0.57–11.99%, respectively. The Lp(a) cholesterol levels determined by AEX-HPLC were significantly correlated with the amounts of Lp(a) protein measured by an immunoturbidimetry method available commercially (r = 0.9503, P < 0.0001). Taken together, this AEX-HPLC method may be effectively applied to the analysis of serum lipoproteins with high levels of Lp(a).

A molecular mechanism for diacylglycerol-mediated promotion of negative caloric balance.

Aims A substitution of diacylglycerol (DAG) oil for triacylglycerol (TAG) oil in diet has been reported to reduce body fat and body weight, possibly by increasing postprandial energy expenditure (EE). We have previously studied plasma serotonin, which increases EE and exists in the small intestine, in individuals who ingested TAG and DAG oil, and found that DAG ingestion elevates plasma serotonin levels by about 50% compared with TAG ingestion. We studied the molecular mechanisms for DAG-mediated increase in serotonin and EE. Methods We studied effects of 1-monoacylglycerol and 2-monoacylglycerol, distinct digestive products of DAG and TAG, respectively, on serotonin release from the Caco-2 cells (the human intestinal cell line, n = 8). Further, we studied effects of 1- and 2-monoacylglycerol, and serotonin on expression of mRNA associated with β-oxidation, FA metabolism, and thermogenesis, in the Caco-2 cells (n = 5). Results 1-monoacylglycerol (100 μM 1-monooleyl glycerol [1-MOG]) significantly increased serotonin release from the Caco-2 cells compared with 2-monoacylglycerol (100 μM 2-MOG) by 36.6%. Expression of mRNA of acyl-CoA oxidase (ACO), fatty acid translocase (FAT), and uncoupling protein-2 (UCP-2) were significantly higher in 100 μM 1-MOG-treated Caco-2 cells than 100 μM 2-MOG-treated cells by 12.8%, 23.7%, and 35.1%, respectively. Further, expression of mRNA of ACO, medium-chain acyl-CoA dehydrogenase, FAT, and UCP-2 were significantly elevated in serotonin (400 nM)-treated Caco-2 cells compared with cells incubated without serotonin by 28.7%, 30.1%, and 39.2%, respectively. Conclusions Our study demonstrated that 1-monoacylglycerol, a digestive product of DAG, increases serotonin release from the Caco-2 cells, and enhances expression of genes associated with β-oxidation, FA metabolism, and thermogenesis, and that serotonin increases expression of these genes, proposing a novel molecular mechanism for DAG-mediated promotion of negative caloric balance.