Metabolomics:Open Access

Metabolomics is a technique in which the small molecule component from a biological source material is analyzed for changes resulting from some set of test conditions. Liquid chromatography tandem mass spectrometry (LC/MS/MS) methods are commonly used because of the sensitivity and specificity of the data collected. The sensitivity of these methods permit the detection of a large number of small molecules, leading to greater coverage of the biochemical pathways involved in the system being tested. The success of metabolomic studies are partially reliant upon instrumentation, but to what extent? Here we present an evaluation of the analytical attributes of a high resolution accurate mass (HRAM) orbitrap based mass spectrometer compared to a unit mass resolution (UMR) ion-trap mass spectrometer as applied to high-throughput, non-targeted metabolomics. To carry out this evaluation, different sets of samples were analyzed and the data evaluated for analytical performance. Two dilution series of authentic standards demonstrated that the HRAM data stream improved the limit of detection from several fold to several orders of magnitude and showed an increased linear dynamic range of an order of magnitude over the UMR data stream. Analysis of a biological serum sample set demonstrated that the HRAM data stream enabled the detection of 118 additional named/known compounds, leading to the detection of 531 tier 1 and tier 2 identified compounds in human serum, with decreased process variability, increased consistency and accuracy of detection and integration.

AICAR (5-aminoimidazole-4-carboxamide 1-ß-D-ribonucleoside) is known to activate the AMP-activated protein kinase (AMPK) in many cell types, including skeletal muscle. We showed that AICAR activated glucose uptake, fatty acid oxidation, and lactate formation by L6 skeletal muscle cell cultures. All of the actions were duplicated by adiponectin, another known activator of AMPK, demonstrating selectivity of AICAR. Moreover, AICAR and adiponectin similarly caused phosphorylation of AMPK, and its target proteins acetyl CoA carboxylase and S6Kinase. While creatine treatment of cells is known to greatly enhance creatine and creatine phosphate levels, it was proposed to paradoxically activate AMPK, and yet not affect glucose transport. However, we found creatine treatment to be without effect on glucose transport, fatty acid oxidation, or phosphorylation of AMPK or its target proteins. Unlike AICAR and adiponectin, creatine treatment stimulated glycogen formation from glucose and inhibited lactate formation. Consistent with this, we observed a diminution in phosphofructokinase expression. Thus, we conclude that creatine and creatine phosphate are not significant allosteric modulators of AMPK in intact cells. We investigated the role of AMPK in glycogen metabolism, using an inhibitor of glycogen phosphorylase. While the inhibitor enhanced glucose incorporation into glycogen, AICAR and adiponectin still diminished glycogen formation, supporting a direct action of AMPK on glycogen synthase. To probe the mechanism for adiponectin, we used siRNA constructs against adenyate kinase and LKB1 (the AMPK kinase). We concluded that adiponectin likely acts through an activation of LKB1, rather than indirectly increasing the turnover of ATP, involving adenylate kinase.

Metabolic consequences of diarrhea-induced sepsis revealed by plasma 1H-Nuclear Magnetic Resonance (NMR) quantitative metabolomics. Diarrhea and sepsis is generally the most common cause of morbidity and mortality in pre-weaned calves. Traditional biomarkers for sepsis are mainly derived from host immune/inflammatory response. Various high-throughput omics technologies facilitate comprehensive screening of sepsis-specific biomarkers in human medicine. The aim of this first study was to reveal the new potential biomarkers in diarrhea-induced septic neonatal calves evaluating by 1H-NMR based quantitative metabolomics. Clinical and laboratory data revealed all the ill calves had leukocytosis, metabolic acidosis and failure of colostral passive transfer. This study produced quantitative data sets that presented differences between patients with diarrhea–induced sepsis and healthy subjects in the level of the water and lipid soluble metabolites. All the lipid soluble metabolites (e.g., sphingomyeline, various fatty acids, etc.) were significantly decreased in diseased calves. Changes in water soluble metabolites (increases in niacinamide, choline + phosphocholine, 2-methylglutarate and isopropanol, and decreases in formate, lysine-arginine, acetate, creatine) were meaningful for pathogenic mechanisms of sepsis. This pilot study showed the implementation of plasma 1H-NMR quantitative metabolomics because it produced a physiologically relevant metabolite data set that distinguished diarrhea-induced sepsis from healthy ones. The metabolites identified and quantified in the study may be new potential biomarkers for systemic inflammatory response syndrome in calf sepsis.

In obese individuals, a high efflux of glycerol from accumulated fat in adipose tissue into the liver is known to be associated with the development of type 2 diabetes. Aquaporin 9 (AQP9) is an aquaglyceroporin which serves as the primary route of hepatic glycerol uptake for gluconeogenesis. Hence, development of AQP9 blockers/regulators may be of potential benefit in controlling hyperglycaemia especially in obese and diabetic individuals. HTS13286 was recently identified as a specific AQP9 inhibitor, even so its limited solubility renders the molecule unsuitable for in vivo application. microRNAs are naturally occurring gene regulators that are often associated with various diseases. The feasibility in selective modulation of microRNAs has introduced a new paradigm for therapeutic applications. In this study, we explore the possibility of using microRNAs to regulate AQP9 expression and eventually glycerolbased gluconeogenesis. In silico prediction of microRNAs targeting the 3’ untranslated region of AQP9 was conducted using miRWalk database. Among the list of potential microRNAs, miR-22 and miR-23a were shortlisted for their high expression in liver and further confirmed to interact with AQP9 via luciferase assay. Over-expression of miR-22 or miR- 23a was able to reduce AQP9 expression (mRNA and protein) and ultimately inhibit glycerol-based gluconeogenesis in HepG2 cells. Livers of diabetic rats were also observed to exhibit an inverse correlation between miR-22, miR-23a and AQP9 expression. As negative modulators of AQP9 expression, miR-22 and miR-23a suggest a potential role in regulating glycerol entry into hepatocytes which could be beneficial in managing glycerol-dependent hyperglycaemic conditions.

Melanoma is a malignant tumor of melanocytes with high capability of invasion and rapid metastasis to other organs.Malignant melanoma is the most common metastatic malignancy found in Gastrointestinal Tract (GI). In this work, the 1H NMR-based metabolomics approach is used to investigate the metabolite profile differences of stomach tissue extracts of metastatic B16-F10 melanoma and control groups in C57BL/6J mouse and to search for specific metabolite biomarker candidates. Principal Component Analysis (PCA), an unsupervised multivariate data analysis method, is used to detect possible outliers, while Orthogonal Projection to Latent Structure (OPLS), a supervised multivariate data analysis method, is employed to evaluate important metabolites responsible for discriminating the control and the melanoma groups. Both PCA and OPLS results reveal that the melanoma group can be well separated from its control group. Among the 50 identified metabolites, it is found that the concentrations of 19 metabolites are significantly changed with the levels of O-phosphocholine and hypoxanthine down-regulated while the levels of isoleucine, leucine, valine, isobutyrate, threonine, cadaverine, alanine, glutamate, glutamine, methionine, citrate, asparagine, tryptophan, glycine, serine, uracil, and formate up-regulated in the melanoma group. These significantly changed metabolites are associated with multiple biological pathways and may be potential biomarkers for metastatic melanoma in stomach.