An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling

Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.     

Phosphorylcholine-containing lipid molecular species profiling in biological tissue using a fast HPLC/QqQ-MS method

A fast reversed-phase liquid chromatography-electrospray ionization triple quadrupole mass spectrometry method was developed for the molecular species profiling of glycerophosphocholine (GPC) and sphingomyelin (SM) in total lipid extracts. A two-stage mass spectrometry strategy was adopted to analyze in detail the composition of lipid molecular species. Precursor ion analysis was first conducted to obtain the preliminary composition profile of the phosphorylcholine-containing lipid. The product ion spectra were sequentially acquired for each recorded signal to determine the molecular structure of the lipid. A total of 150 GPCs and 12 SMs were identified in the fetal mouse lung with relative amounts ranging from 13.7?% to less than 0.002?% (normalizing by the total signal response). A column packed with core–shell particles was used to obtain excellent chromatographic separation with a shorter time demand in a conventional high-performance liquid chromatography system. Considering the compromise between the chromatographic efficiency and the electrospray signal response, the optimization of the mobile phase improves the chromatographic plate number to approximately 40,000 and the detection limits to less than 0.001?mg/L. The applicability of the method was validated through a study of chemically induced early lung maturation. The metabolic alteration in the fetal mouse lung was clearly reflected in the GPC and SM composition with several characteristics of the molecular structure that related to the character of the phospholipid layer upon the epithelial lining of alveoli and the relevant cell function. The results indicated that this analytical strategy is reliable for comprehensive molecular species profiling of GPC and SM and might be extended to the analysis of other phospholipids.

Accelerated,untargeted metabolomics analysis of cutaneous T‐cell lymphoma reveals metabolic shifts in plasma and tumor adjacent skins of xenograft mice

Cutaneous T‐cell lymphoma (CTCL) is a heterogeneous group of skin‐homing T‐cell neoplasms. Clinical management is stage based but diagnosis and prognosis could be extremely challenging. The presented study aims to explore the metabolic profiling of CTCL by an accelerated untargeted metabolomics data analysis tool “Mummichog” to facilitate the discoveries of potential biomarkers for clinical early stage diagnosis, prognosis, and treatments in CTCL. Ultra high‐performance liquid chromatography–quadrupole time‐of‐flight–based untargeted metabolomics were conducted on the skin and plasma of CTCL mice. It showed that the metabolism of skin changed greatly versus control samples in the development of CTCL. Increased l ‐glutamate and decreased adenosine monophosphate were the most essential metabolic features of CTCL tumor and tumor adjacent skins. Unique metabolism changes in tumor adjacent non‐involved skin tissues (ANIT) occurred in the progress of carcinogenesis, including upregulated cytidine‐5′‐triphosphate, aberrant biosynthesis of prostaglandins, pyrimidine, mevalonate pathway, and tryptophan degradation. Sharply elevated 5‐phospho‐α‐d ‐ribose 1‐diphosphate (PRPP) marked the final state of tumor in CTCL. In the plasma, systematic shifts in corticosterone, sphingolipid, and ceramide metabolism were found. These uncovered aberrant metabolites and metabolic pathways suggested that the metabolic reprogramming of PRPP in tumor tissues may cause the disturbance of cytidine and uridine metabolic homeostasis in ANIT. Accumulative cytidine‐5′‐triphosphate in ANIT may exert positive feedback on the PRPP level and leads to CTCL further development. In addition, the accelerated data analysis tool “Mummichog” showed good practicability and can be widely used in high‐resolution liquid chromatography mass spectrometry–based untargeted metabolomics.     

LC Coupled with TOFMS for Metabonomics Study of Mini-pigs with Atherosclerosis

In this study, serum metabolic profiles of mini-pigs with atherosclerosis (AS) were analyzed by LC–TOFMS. Partial least-squares to latent structure-discriminant analysis and orthogonal projection to latent structure-discriminant analysis were used for group differentiation and selection of potential biomarkers. The mini-pig disease models were constructed by feeding a high-fat diet and inducing coronary injury, in accordance with the mechanism of AS pathogenesis. To characterize the development of AS, serum samples were collected and analyzed at two time points (two and ten weeks). Separate distinct clustering of results from normal and model mini-pigs could be observed for both the two and ten-week samples. With the development of AS, the metabolism of the model mini-pigs was more substantially disturbed. Major metabolites contributing to the discrimination were fatty acids, lysophosphatidylcholines, and bile acids. These potential biomarkers are related with inflammation, oxidative stress, and abnormal lipid and energy metabolism.

     

N-苄基十六碳酰胺促小鼠Leydig细胞增殖和分泌睾酮的1H NMR代谢组学研究

采用基于核磁共振氢谱(¹H NMR)的细胞代谢组学技术探讨了玛咖有效成分N-苄基十六碳酰胺(NBH)促进小鼠Leydig细胞增殖和分泌睾酮的作用机制. 测定了小鼠Leydig细胞在给药前后的细胞增殖率和细胞培养液中的睾酮含量, 采用主成分分析和正交偏最小二乘判别分析, 研究了小鼠Leydig细胞在给药前后细胞破碎液中的代谢物差异, 并进行了代谢通路分析. 实验结果表明, NBH能提高小鼠Leydig细胞增殖率和睾酮分泌量, 给药后小鼠Leydig细胞破碎液中的代谢轮廓明显改变, 共鉴定亮氨酸、 赖氨酸、 鲨肌醇、 缬氨酸和丙氨酸等24种差异代谢物. 经Metaboanalyst分析发现, 差异代谢物主要涉及丙氨酸/天冬氨酸/谷氨酸代谢、 甘氨酸/丝氨酸/苏氨酸代谢、 谷胱甘肽代谢及丙酮酸代谢等10条新陈代谢和遗传信息处理代谢通路, 初步阐明了NBH通过调节上述代谢通路的相关节点发挥促进小鼠Leydig细胞增殖和分泌睾酮作用.     

Chromatographic lipid profiling of stress-exposed cells

Lipidomics is an emerging field of science not only due to its integral part of cell biology and biophysics but also due to the key role of lipids in the modulation of membrane physical properties, signaling, and cell death regulation. The aim of this study was to characterize changes in N-palmitoyl ceramide concentration and in the global lipid profile in macrophages challenged by oxidized low-density lipoprotein and nutrient deprived hepatocytes. For this purpose, a quantitative targeted method based on gas chromatography-mass spectrometry for the determination of total N-palmitoyl ceramide concentrations in the cellular membranes of cells under stress was used. Ultrahigh-performance liquid chromatography-quadrupole-time of flight mass spectrometry was applied for the comprehensive profiling of lipids. In essence, we found that both models of cellular stress caused an increase in N-palmitoyl ceramide levels. In addition, increased levels of other ceramides were observed as well as up- and down-regulation of several other lipid species.     

Integrated metabolomics analysis of the effect of PPARδ agonist GW501516 on catabolism of BCAAs and carboxylic acids in diabetic mice

The peroxisome proliferator-activated receptor(PPARδ) agonists are reported to improve insulin sensitivity,reduce glucose levels,and alleviate dysfunctional lipid metabolism in animal models of type 2 diabetes mellitus.However,the underlying mechanisms remain incompletely understood.Metabolism plays an essential role in the biological system.Monitoring of metabolic changes in response to disease conditions or drug treatment is critical for better understanding of the pathophysiological mechanisms.In this study,metabolic profiling analysis by gas chromatography-mass spectrometry integrated with targeted analysis by liquid chro matography-mass spectrometry was carried out in plasma samples of db/db diabetic mice after six-week treatment of PPARδ agonist GW501516.GW501516 treatment significantly altered levels of metabolites,such as branched-chain amino acids(BCAAs),BCAA metabolites(3-hydroxyisobutyric acid and 3-hydroxyisovaleric acid),long-chain fatty acids,uric acid and ketone bodies(3-hydroxybutyric acid and 2-hydroxybutyric acid) which are all associated with the impaired systemic insulin sensitivity.The pre sent results indicate the beneficial effect of PPARδ agonist in alleviating insulin resistance of diabetic mice by favorably modulating metabolic profile,thus providing valuable information in understanding the therapeutic potential of PPARδ agonists in correcting metabolic dysfunction in diabetes.     

Comparative evaluation of labelling patterns and turnover of lipids,tagged by 15 /p-123I-phenyl-/ pentadecanoic and 1-14C-palmitic acid

Uptake and turnover of chloroform/methanol extractable tissue lipids labelled in vivo simultaneously with 15/p-¹²³I-phenyl-/pentadecanoic and l-¹⁴C-palmitic acid were compared. Lipid turnover studies were performed in fasted pentobarbital-anaesthetized Wistar rats in tissues with highly varying free fatty acid turnover rates. In all tissues investigated, i.e. heart, lung, liver, spleen and kidneys, both tracers labelled nearly identical lipid fraction. Main tracer uptake was found in free fatty acids, phospholipids, diglycerides and triglycerides. A highly significant correlation of uptake and turnover in main tissue lipid fraction indicated an essentially identical metabolic pathway of both tracers in intermediary tissue lipid metabolism. Concordant tracer uptake and turnover patterns in tissue of lipids with highly varying free fatty acid metabolic rates suggested that intrinsic metabolic activity of the tissue and respective lipid fraction was the major determinant of metabolic handling of both iodophenyl fatty- and palmitic acid. Thus, the feasibility of iodophenylpentadecanoic acid as free fatty acid tracer for studying tissue lipid metabolism is demonstrated.     

Metabolomic profiling of rat serum associated with isoproterenol-induced myocardial infarction using ultra-performance liquid chromatography/time-of-flight mass spectrometry and multivariate analysis

An ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS)-based metabolomic approach was developed to characterize the metabolic profile associated with isoproterenol (ISO)-induced myocardial infarction (MI). Analysis of the serum samples revealed distinct changes in the biochemical patterns of ISO-induced rats. A multivariate statistical method, supervised partial least squares-discriminant analysis (PLS-DA), was then used for screening of potential biomarkers. As a result, 13 lipid biomarkers, including lysophosphatidylcholines (Lyso-PCs) and fatty acids were identified by the accurate mass measurement of TOF-MS. The relationship between abnormal lipid metabolism and the formation of MI were also studied. This work demonstrates the utility of UPLC/TOF-MS-based metabolic profiling combined with multivariate analysis as a powerful tool to further investigate the pathogenesis of cardiovascular diseases.     

Quantitative analysis of urinary endogenous markers for the treatment effect of Radix Scutellariae on type 2 diabetes rats

The effect of Radix Scutellariae treated on type 2 diabetic rats has been investigated by a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based urinary quantitative approach. In this research, multiple reactions monitoring mode of MS/MS in LC-MS/MS analysis was used to quantitatively analyze the concentrations of 7 endogenous compounds in urine of normal control group, type 2 diabetic model group and Radix Scutellariae-treated group, and multivariate statistical analysis was utilized for MS data processing. The above-mentioned three groups can be distinguished via pattern recognition. The obtained results indicated that Radix Scutellariae affect the urinary metabolic profiling of type 2 diabetic rats on the polyol pathway, protein glycation reaction and amino acids metabolism pathway. According to these results, Radix Scutellariae should have the pharmacological effect on preventing or delaying the onset and progression of diabetes and its complications.     

Biomass Composition, Lipid Characterization, and Metabolic Profile Analysis of the Fed-Batch Fermentation Process of Two Different Docosahexanoic Acid Producing Schizochytrium sp. Strains

Growth and fermentation characteristics, biomass composition, lipid characterization and metabolic profiling analysis of two different Schizochytrium sp. strains, the original strain and the industrial adaptive strain, were investigated in the fed-batch fermentation process. The final cell biomass, total lipids content, docosahexanoic acid (DHA) content and DHA productivity of the adaptive strain were much higher than those of the original strain. The metabolic distinctions which extensively existed between these two strains were revealed by the score plot of principal component analysis. In addition, potential biomarkers responsible for discriminating different strains were identified as myo-inositol, histidine, alanine, asparagine, cysteine, and oxalic acid. These findings provided new insights into the industrial strain screening and further improvement of DHA production by Schizochytrium sp.     

基于液相色谱-质谱联用技术的代谢组学方法用于中药通心络和人参对过度疲劳大鼠干预作用的评价

用代谢组学方法评价了中药通心络和人参对过度疲劳大鼠的干预作用.通过构造大鼠过度疲劳模型,并分别用通心络和人参进行干预,采用快速液相色谱-离子阱飞行时间质谱(UFLC-IT-TOF-MS)获取大鼠血浆代谢轮廓,并用正交偏最小二乘法(OPLS)进行多变量统计分析,分别找出用于区分通心络和人参干预组大鼠同正常对照大鼠、过度疲...     

Ceramide-enriched microdomains in planar membranes

Ceramide has a large effect on the properties of biological membranes, increasing lipid order and promoting lateral phase separation, and plays an important role in cell signaling. This review provides an overview of recent studies of the effects of direct ceramide incorporation and enzymatic ceramide generation on planar supported membranes, including lipid monolayers and supported lipid bilayers. Recent studies have focused on understanding the nucleation, growth and morphology of ceramide gel domains, characterizing the properties of ceramide-rich membrane phases and investigating the effects of ceramide on phase-separated membranes with co-existing liquid-ordered and fluid phases, as models for cellular membranes.     

Profiling changes triggered during maturation of dendritic cells: a lipidomic approach

Lipids are important in several biological processes because they act as signalling and regulating molecules, or, locally, as membrane components that modulate protein function. This paper reports the pattern of lipid composition of dendritic cells (DCs), a cell type of critical importance in inflammatory and immune responses. After activation by antigens, DCs undergo drastic phenotypical and functional transformations, in a process known as maturation. To better characterize this process, changes of lipid profile were evaluated by use of a lipidomic approach. As an experimental model of DCs, we used a foetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). The results showed that LPS treatment increased ceramide (Cer) and phosphatidylcholine (PC) levels and reduced sphingomyelin (SM) and phosphatidylinositol (PI) content. Mass spectrometric analysis of a total lipid extract and of each class of lipids revealed that maturation promoted clear changes in ceramide profile. Quantitative analysis enabled identification of an increase in the total ceramide content and enhanced Cer at m/z 646.6, identified as Cer(d18:1/24:1), and at m/z 648.6, identified as Cer(d18:1/24:0). The pattern of change of these lipids give an extremely rich source of data for evaluating modulation of specific lipid species triggered during DC maturation.     

基于超高效液相色谱-四极杆飞行时间质谱的缺血性脑卒中的代谢组学研究

采用基于超高效液相色谱-四极杆飞行时间质谱的代谢组学方法,研究缺血性脑卒中患者和健康人群的血浆,分析了缺血性脑卒中的生物标志物.实验收集30例缺血性脑卒中患者和17例健康志愿者的血浆样品,采用主成分分析和正交偏最小二乘判别分析,研究了缺血性脑卒中患者组和健康对照组血浆中的代谢物差异,并进行了代谢通路分析.实验结果表明,患者组血浆中的二氢神经鞘氨醇、植物鞘氨醇等物质含量升高,谷氨酰胺、焦谷氨酸和2-酮丁酸等物质含量降低.结果表明,脑卒中不仅影响了鞘脂类和氨基酸的代谢,还对能量代谢产生了显著的影响.     

基于超高效液相色谱-串联质谱的股骨头坏死组织外泌体脂质代谢组学分析

股骨头坏死(ONFH)是一种可导致股骨头塌陷进而需要接受全髋关节置换的疾病.外泌体作为一种细胞间交流的方式,在一系列生理和病理过程中起着至关重要的作用,已在疾病的诊断和治疗中发挥独特作用.该研究利用非靶向代谢组学方法,探讨股骨头坏死组织外泌体内的脂质代谢特征,阐释股骨头坏死时机体发生的脂质代谢变化.该研究采用超速离心的...     

基于液相色谱-质谱联用的代谢组学技术研究Pokemon诱导的胞内脂代谢变化

Pokemon是一种转录抑制因子,能够通过影响染色质的重组或直接与抑癌基因结合而抑制抑癌基因的转录,促使肿瘤形成。该文利用基于液相色谱-质谱联用的代谢组学技术研究了Pokemon在肝癌中调控细胞代谢的作用机制。通过脂质转染,获得了Pokemon高表达的HL7702细胞,分别收集转染后不同时间点的细胞。利用基于液相色谱-质谱联用技术的代谢组学方法,分析胞内代谢物的成分。根据多元统计分析的结果选出差异显著的候选代谢物,通过数据库（METLIN和HMDB）检索、二级图谱比对进行结构解析,确证了36种代谢物。通过KEGG数据库检索发现这些代谢物主要与脂质合成相关。进一步分析发现脂质合成途径中乙酰辅酶羧化酶和脂肪酸合成酶均被激活。结果显示,Pokemon可通过激活细胞中脂质合成通路而影响细胞的代谢。     

Metabolic profiling study on potential toxicity and immunotoxicity‐biomarker discovery in rats treated with cyclophosphamide using HPLC‐ESI‐IT‐TOF‐MS

Despite the recent advances in understanding toxicity mechanism of cyclophosphamide (CTX), the development of biomarkers is still essential. CTX‐induced immunotoxicity in rats by a metabonomics approach was investigated using high‐performance liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry (HPLC‐ESI‐IT‐TOF‐MS). The rats were orally administered CTX (30 mg/kg/day) for five consecutive days, and on the fifth day samples of urine, thymus and spleen were collected and analyzed. A significant difference in metabolic profiling was observed between the CTX‐treated group and the control group by partial least squares‐discriminant analysis (PLS‐DA), which indicated that metabolic disturbances of immunotoxicity in CTX‐treated rats had occurred. One potential biomarker in spleen, three in urine and three in thymus were identified. It is suggested that the CTX‐toxicity mechanism may involve the modulation of tryptophan metabolism, phospholipid metabolism and energy metabolism. This research can help to elucidate the CTX‐influenced pathways at a low dose and can further help to indicate the patients' pathological status at earlier stages of toxicological progression after drug administration. Copyright © 2014 John Wiley & Sons, Ltd.