7-Ketocholesterol Induces Lipid Metabolic Reprogramming and Enhances Cholesterol Ester Accumulation in Cardiac Cells.

7-Ketocholesterol (7KCh) is a major oxidized cholesterol product abundant in lipoprotein deposits and atherosclerotic plaques. Our previous study has shown that 7KCh accumulates in erythrocytes of heart failure patients, and further investigation centered on how 7KCh may affect metabolism in cardiomyocytes. We applied metabolomics to study the metabolic changes in cardiac cell line HL-1 after treatment with 7KCh. Mevalonic acid (MVA) pathway-derived metabolites, such as farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate, phospholipids, and triacylglycerols levels significantly declined, while the levels of lysophospholipids, such as lysophosphatidylcholines (lysoPCs) and lysophosphatidylethanolamines (lysoPEs), considerably increased in 7KCh-treated cells. Furthermore, the cholesterol content showed no significant change, but the production of cholesteryl esters was enhanced in the treated cells. To explore the possible mechanisms, we applied mRNA-sequencing (mRNA-seq) to study genes differentially expressed in 7KCh-treated cells. The transcriptomic analysis revealed that genes involved in lipid metabolic processes, including MVA biosynthesis and cholesterol transport and esterification, were differentially expressed in treated cells. Integrated analysis of both metabolomic and transcriptomic data suggests that 7KCh induces cholesteryl ester accumulation and reprogramming of lipid metabolism through altered transcription of such genes as sterol O-acyltransferase- and phospholipase A2-encoding genes. The 7KCh-induced reprogramming of lipid metabolism in cardiac cells may be implicated in the pathogenesis of cardiovascular diseases.

Characteristic of Metabolic Status in Heart Failure and Its Impact in Outcome Perspective.

Metabolic alterations have been documented in peripheral tissues in heart failure (HF). Outcomes might be improved by early identification of risk. However, the prognostic information offered is still far from enough. We hypothesized that plasma metabolic profiling potentially provides risk stratification for HF patients. Of 61 patients hospitalized due to acute decompensated HF, 31 developed HF-related events in one year after discharge (Event group), and the other 30 patients did not (Non-event group). The plasma collected during hospital admission was analyzed by an ultra-high performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS)-based metabolomic approach. The orthogonal projection to latent structure discriminant analysis (OPLS-DA) reveals that the metabolomics profile is able to distinguish between events in HF. Levels of 19 metabolites including acylcarnitines, lysophospholipids, dimethylxanthine, dimethyluric acid, tryptophan, phenylacetylglutamine, and hypoxanthine are significantly different between patients with and without event (p < 0.05). Established risk prediction models of event patients by using receiver operating characteristics analysis reveal that the combination of tetradecenoylcarnitine, dimethylxanthine, phenylacetylglutamine, and hypoxanthine has better discrimination than B-type natriuretic peptide (BNP) (AUC 0.871 and 0.602, respectively). These findings suggest that metabolomics-derived metabolic profiling have the potential of identifying patients with high risk of HF-related events and provide insights related to HF outcome.

Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

Enhancement of the stability of chlorophyll using chlorophyll-encapsulated polycaprolactone microparticles based on droplet microfluidics.

Chlorophyll is a valuable bioactive compound, which is used as a natural food coloring agent and a photosensitizer for photodynamic therapy because of its antioxidant properties, antimutagenic ability, and near-infrared fluorescence. However, chlorophyll is unstable when it comes to retaining its antioxidant activity, when exposed to oxygen, high temperature, or light environments. To enhance the stability of chlorophyll, a polymer encapsulation method was proposed. Polycaprolactone (PCL) was employed to encapsulate the chlorophyll, and the particles size of the composites was controlled through droplet microfluidics. The composites (chlorophyll-encapsulated PCL particles) were characterized through UV-VIS spectrometry, SEM, optical microscopy, and light exposure. The particles were spherical, with diameters adjustable from 68 to 247 µm. Additionally, the chlorophyll-encapsulated PCL particles exhibited considerably prolonged chlorophyll stability. The solid microparticle is more convenient for storage and transportation, and have great potential for application in the food industry.

Sedoheptulose-1,7-bisphospate Accumulation and Metabolic Anomalies in Hepatoma Cells Exposed to Oxidative Stress.

We have previously shown that GSH depletion alters global metabolism of cells. In the present study, we applied a metabolomic approach for studying the early changes in metabolism in hydrogen peroxide- (H2O2-) treated hepatoma cells which were destined to die. Levels of fructose 1,6-bisphosphate and an unusual metabolite, sedoheptulose 1,7-bisphosphate (S-1,7-BP), were elevated in hepatoma Hep G2 cells. Deficiency in G6PD activity significantly reduced S-1,7-BP formation, suggesting that S-1,7-BP is formed in the pentose phosphate pathway as a response to oxidative stress. Additionally, H2O2 treatment significantly increased the level of nicotinamide adenine dinucleotide phosphate (NADP+) and reduced the levels of ATP and NAD+. Severe depletion of ATP and NAD+ in H2O2-treated Hep G2 cells was associated with cell death. Inhibition of PARP-mediated NAD+ depletion partially protected cells from death. Comparison of metabolite profiles of G6PD-deficient cells and their normal counterparts revealed that changes in GSH and GSSG per se do not cause cell death. These findings suggest that the failure of hepatoma cells to maintain energy metabolism in the midst of oxidative stress may cause cell death.

Disturbance of Plasma Lipid Metabolic Profile in Guillain-Barre Syndrome.

Guillain-Barre Syndrome (GBS) is an inflammatory disease of the peripheral nervous system. Given that plasma metabolic profiles in GBS patients have never been explored, plasma samples of 38 GBS patients, 22 multiple sclerosis (MS) patients, and 40 healthy controls were analyzed by using untargeted and targeted metabolomics analysis. The untargeted analysis showed that levels of a set of plasma lipid metabolites were significantly decreased in GBS patients compared to the controls. Furthermore, the targeted analysis demonstrated that levels of 41 metabolites in GBS patients were significantly changed compared to either the controls or MS patients. A further metabolic analysis showed that 12 of 41 metabolites were significantly lower in classical GBS patients compared to Miller-Fisher syndrome. Among them, each of PCae C34:0, PCae C42:2, PCae C42:3, and SM C24:0 was inversely correlated with Hughes functional grading scale of GBS patients at both nadir and discharge. Receiver operating characteristic curve analysis of combination of three metabolites (PCaa C42:2, PCae C36:0 and SM C24:0) showed a good discrimination between the GBS and the controls (area under curve = 0.86). This study has demonstrated disruption of lipid metabolites in GBS may be potential biomarkers to indicate disease severity and prognosis of GBS.

Increased p-cresyl sulfate level is independently associated with poor outcomes in patients with heart failure.

Amino acid-derived metabolites, including protein-bound uremic toxins, may have prognostic value for patients with heart failure (HF). The aim of this study was to investigate whether p-cresyl sulfate (PCS), indoxyl sulfate (IS), and arginine metabolites provided prognostic values in addition to the traditional biomarker, B-type natriuretic peptide (BNP), in patients with HF. Chromatography mass spectrometry was performed to measure tyrosine, tryptophan, arginine, PCS, IS, and asymmetric (ADMA) and symmetric dimethylarginine (SDMA) in the plasma from 51 normal controls and 136 HF patients. Compared to the normal controls, PCS levels significantly increased in HF patients (p = 0.003). During the follow-up (2.3 ± 1.1 years), 35 (25.7 %) patients experienced a composite event of death or HF-related re-hospitalization. In univariable analysis, PCS, estimated glomerular filtration rate (eGFR), BNP, DMA/arginine ratio, and ADMA/arginine ratio were associated with a higher rate of composite events. In the multivariable analysis, PCS was the only independent predictor of composite events [hazard ratio (HR) 1.06 (per 10 µM), 95 % confidence interval (CI) 1.01-1.11, p = 0.02]. Kaplan-Meier curves showed that a PCS level of ≥50 µM was significantly associated with a higher composite event rate than those with a PCS level of <50 µM (Log rank = 5.11, p = 0.024; HR 2.13, 95 % CI 1.09-4.16, p = 0.02). In conclusion, among protein-bound uremic toxins, eGFR, and DMA metabolites, increased PCS is the only independent predictor of HF-related events in patients with HF. A combination of PCS and BNP should better risk-stratify patients with HF.

Metabolic disturbances identified in plasma are associated with outcomes in patients with heart failure: diagnostic and prognostic value of metabolomics.

BACKGROUND: Identification of novel biomarkers is needed to improve the diagnosis and prognosis of heart failure (HF). Metabolic disturbance is remarkable in patients with HF. OBJECTIVES: This study sought to assess the diagnostic and prognostic values of metabolomics in HF. METHODS: Mass spectrometry-based profiling of plasma metabolites was performed in 515 participants; the discovery phase study enrolled 51 normal control subjects and 183 HF patients, and the validation study enrolled 63 control subjects and 218 patients with stage C HF. Another independent group of 32 patients with stage C HF who recovered to New York Heart Association functional class I at 6 and 12 months was profiled as the "recovery" group. RESULTS: A panel of metabolites, including histidine, phenylalanine, spermidine, and phosphatidylcholine C34:4, has a diagnostic value similar to B-type natriuretic peptide (BNP). In the recovery group, the values of this panel significantly improved at 6 and 12 months. To evaluate the prognostic values, events were defined as the combined endpoints of death or HF-related re-hospitalization. A metabolite panel, which consisted of the asymmetric methylarginine/arginine ratio, butyrylcarnitine, spermidine, and the total amount of essential amino acids, provided significant prognostic values (p < 0.0001) independent of BNP and traditional risk factors. The prognostic value of the metabolite panel was better than that of BNP (area under the curve of 0.85 vs. 0.74 for BNP) and Kaplan-Meier curves (log rank: 17.5 vs. 9.95). These findings were corroborated in the validation study. CONCLUSIONS: Metabolomics demonstrate powerful diagnostic value in estimating HF-related metabolic disturbance. The profile of metabolites provides better prognostic value versus conventional biomarkers.

The role of heat shock protein 70 in the protective effect of YC-1 on ß-amyloid-induced toxicity in differentiated PC12 cells.

Neurodegenerative brain disorders such as Alzheimer's disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aß25-35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aß25-35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in µ-calpain expression. Moreover, Aß25-35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5-10 µM) prevented the cell death induced by Aß25-35. In addition, YC-1 (1, 10 µM) significantly blocked Aß25-35-induced µ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5-20 µM) alone or combined with Aß25-35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aß25-35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aß25-35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.

Retrospectively comparative evaluation of the first- and second-line chemotherapy with campto and oxaliplatin combined with oral tegafur/uracil (UFT)/leucovorin (LV) in patients with metastatic colorectal cancer.

Oral tegafur/uracil (UFT), like an intravenous drip with 5-fluorouracil (5-FU)/leucovorin (LV), has been regarded as an active regimen in the treatment of advanced colorectal cancer. In this clinical trial, we evaluate the toxicity and efficacy of regimens containing oral UFT/LV, instead of an intravenous drip with 5-FU/LV, in the treatment of metastatic colorectal cancer. A phase II study involving 39 patients with metastatic colorectal cancer (CRC) who received UFT/LV plus irinotecan (Group A)/oxaliplatin (Group B) alternated with UFT/LV plus oxaliplatin (Group A)/irinotecan (Group B) was evaluated. The overall tumor control rate (CR + PR + SD) was approximately 64%, and median overall survival was approximately 12 months. Of the 547 doses the 39 patients received, 6 events (1.10%) of neutropenia, 5 events (0.91%) of diarrhea, 2 events (0.37%) of stomatitis, and 2 events (0.37%) of anemia were observed. The alternating regimen seems to be effective and well tolerated for patients with metastatic CRC.

Clinical relevance of plasma homocysteine levels in Taiwanese patients with coronary artery disease.

Homocysteine (Hcy) has recently been considered as a risk factor for cardiovascular disease. Coronary artery disease (CAD) is the third most common cause of mortality in Taiwan. The objective of our present study is to delineate the relationship between the plasma Hcy level and CAD in Taiwanese using an improved enzymatic method. Blood samples were collected from 86 CAD patients, which include 38 patients with acute coronary syndrome (ACS) and 48 patients with stable CAD, and 89 controls. Plasma Hcy levels were measured by HPLC and enzymatic methods. Plasma Hcy levels were 8.27 +/- 1.74, 8.50 +/-1.88 and 11.76 +/- 4.58 muM in normal controls, patients with stable CAD and those with ACS, respectively. Plasma Hcy levels were elevated in the cohort of ACS patients, as compared with those of normal controls or patients with stable CAD. Within the CAD group, patients with the highest quartile Hcy level (10.18-23.73 muM) had a significantly higher odd ratio for ACS, as compared with those with the lowest quartile Hcy level after multivariate adjustment. Plasma Hcy concentration can be used as an independent risk factor of ACS, particularly for those with a history of CAD. Moreover, our improved enzymatic assay can be automated for large-scale screening of high-risk cohorts.

Use of gain-of-function study to delineate the roles of crumbs in Drosophila eye development.

The cell polarity gene, crumbs (crb)), has been shown to participate in the development and degeneration of the Drosophila retina. Mutations in CRB1, the human homologue of Drosophila crb, also result in retinitis pigmentosa and Leber congenital amaurosis. In this study, we used the gain-of-function approach to delineate the roles of CRB in developing Drosophila eye. In the third-instar larval stage, eye development is initiated with photoreceptor differentiation and positioning of photoreceptor nuclei in the apical cellular compartment of retinal epithelium. In the pupal stage, differentiated photoreceptors begin to form the photosensitive structures, the rhabdomeres, at their apical surface. Using GMR-Gal4 to drive overexpression of the Crb protein at the third-instar eye disc, we found that differentiation of photoreceptors was disrupted and the nuclei of differentiated photoreceptors failed to occupy the apical compartment. Using HS-Gal4 to drive Crb overexpression in pupal eyes resulted in interference with extension of the adherens junctions and construction of the rhabdomeres, and these defects were stage-dependent. This gain-of-function study has enabled us to delineate the roles of Crb at selective stages of eye development in Drosophila.