Combined transcriptome and metabolome analyses of metformin effects reveal novel links between metabolic networks in steroidogenic systems.

Metformin is an antidiabetic drug, which inhibits mitochondrial respiratory-chain-complex I and thereby seems to affect the cellular metabolism in many ways. It is also used for the treatment of the polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. In addition, metformin possesses antineoplastic properties. Although metformin promotes insulin-sensitivity and ameliorates reproductive abnormalities in PCOS, its exact mechanisms of action remain elusive. Therefore, we studied the transcriptome and the metabolome of metformin in human adrenal H295R cells. Microarray analysis revealed changes in 693 genes after metformin treatment. Using high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS-NMR), we determined 38 intracellular metabolites. With bioinformatic tools we created an integrated pathway analysis to understand different intracellular processes targeted by metformin. Combined metabolomics and transcriptomics data analysis showed that metformin affects a broad range of cellular processes centered on the mitochondrium. Data confirmed several known effects of metformin on glucose and androgen metabolism, which had been identified in clinical and basic studies previously. But more importantly, novel links between the energy metabolism, sex steroid biosynthesis, the cell cycle and the immune system were identified. These omics studies shed light on a complex interplay between metabolic pathways in steroidogenic systems

Lab-on-a-chip for multiplexed biosensing of residual antibiotics in milk

A multiplexed immunoassay-based antibiotic sensing device integrated in a lab-on-a-chip format is described. The approach is multidisciplinary and involves the convergent development of a multi-antibiotic competitive immunoassay based on sensitive wavelength interrogated optical sensor (WIOS) technology and a polymer-based self-contained microfluidic cartridge. Immunoassay solutions are pressure-driven through external and concerted actuation of a single syringe pump and multiposition valve. Moreover, the use of a novel photosensitive material in a one step fabrication process allowed the rapid fabrication of microfluidic components and interconnection port simultaneously. Pre-filled microfluidic cartridges were used as binary response rapid tests for the simultaneous detection of three antibiotic families – sulfonamides, fluoroquinolones and tetracyclines – in raw milk. For test interpretation, any signal lower than the threshold value obtained for the corresponding Maximum Residue Limit (MRL) concentration (100 µg L-1) was considered negative for a given antibiotic. The reliability of the multiplexed detection system was assessed by way of a validation test carried out on a series of six blind milk samples. A test accuracy of 95% was calculated from this experiment. The whole immunoassay procedure is fast (less than 10 minutes) and easy to handle (automated actuation)

Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts.

INTRODUCTION A decline in mitochondrial function represents a key factor of a large number of inborn errors of metabolism, which lead to an extremely heterogeneous group of disorders. OBJECTIVES To gain insight into the biochemical consequences of mitochondrial dysfunction, we performed a metabolic profiling study in human skin fibroblasts using galactose stress medium, which forces cells to rely on mitochondrial metabolism. METHODS Fibroblasts from controls, complex I and pyruvate dehydrogenase (PDH) deficient patients were grown under glucose or galactose culture condition. We investigated extracellular flux using Seahorse XF24 cell analyzer and assessed metabolome fingerprints using NMR spectroscopy. RESULTS Incubation of fibroblasts in galactose leads to an increase in oxygen consumption and decrease in extracellular acidification rate, confirming adaptation to a more aerobic metabolism. NMR allowed rapid profiling of 41 intracellular metabolites and revealed clear separation of mitochondrial defects from controls under galactose using partial least squares discriminant analysis. We found changes in classical markers of mitochondrial metabolic dysfunction, as well as unexpected markers of amino acid and choline metabolism. PDH deficient cell lines showed distinct upregulation of glutaminolytic metabolism and accumulation of branched-chain amino acids, while complex I deficient cell lines were characterized by increased levels in choline metabolites under galactose. CONCLUSION Our results show the relevance of selective culture methods in discriminating normal from metabolic deficient cells. The study indicates that untargeted fingerprinting NMR profiles provide physiological insight on metabolic adaptations and can be used to distinguish cellular metabolic adaptations in PDH and complex I deficient fibroblasts

Correction to: Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts.

The original version of this article contained an error in Table 2. The text in the second header line should read "GAL supernatant" and "GAL Medium" instead of "GLC supernatant" and "GLC Medium". The corrected Table 2 is given below. The original article has been corrected

Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three-dimensional organotypic brain cell cultures

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable

Improving the metabolic stability of cultured cells during extended HR-MAS NMR measurements by prior heating

Introduction: 1HHRMAS NMR is an established tool for metabolic characterization of biological samples. However, the accuracy of biomarker detection depends on the sample stability over measurement time. Previously, Duarte et al. found enhanced metabolite visibility and different lipid profiles in lysed compared to frozen cells [1]. Here we investigated effects of shorttime heating prior to HRMAS measurements on metabolite stability of cells. We hypothesized that cell heating has only minor effects on initial metabolite contents (i.e. initially similar spectra with and without heating) and results in increased temporal metabolite stability due to reduced enzymatic activity. Methods: The metabolite content of six lysed nonheated (LFB) and six lysed heated fibroblastsamples (LHFB) was measured as a function of time. Additionally, one sample each of nonheated and heated lysed adrenal cells (LAD & LHAD), which are less robust than fibroblasts, were measured. After lysis according to [1], half of the samples were heated (70°C) for 20min. 1HPROJECT [2] spectra were obtained on a Bruker AvanceII spectrometer (500.13MHz, 277K, MAS=3kHz) at different times over 9 hours. Individual peak analysis was performed investigating temporal changes. For fibroblasts, statistical methods were applied including PCA. Results and Discussion: PCA results and individual peak analysis clearly confirmed our hypothesis: The temporal metabolite stability for LHFB was largely maintained in contrast to LFB. Average temporal variation of all peaks was 14.4% for LHFB compared to 43.1% for LFB. Additionally, the PCA plot demonstrated close clustering for LHFB, whereas LFB were spreading out over time. Also LHAD showed temporal metabolic stability, while LAD exhibited strong changes. Our results suggest using cell lysis in combination with heating for extended longterm HRMAS NMR measurements, in order to minimize metabolite content modifications over measurement time. References: 1. Duarte IF, et al. Anal. Chem. 2009;81:5023. 2. Aguilar JA, et al. Chemical Communications 2012;48:811

Metabolic stability of cells for extended metabolomical measurements using NMR. A comparison between lysed and additionally heat inactivated cells.

NMR measurements for metabolic characterization of biological samples like cells, biopsies or plasma, may take several hours for advanced methods. Preanalytical issues, such as sample preparation and stability over the measurement time, may have a high impact on metabolite content, and potentially lead to misinterpretation. The aim of this study was therefore to investigate by 1H HR-MAS NMR the impact of different cell handling preparation protocols on the stability of the cell metabolite content over the measurement time. For this purpose, the metabolite content of fibroblasts and adrenal cells were measured at different time points after lysis and after additional heating. Interestingly the results showed similar metabolite concentrations between lysed and lysed-heated cells at the beginning of the measurement, but increasing differences after some hours of measurement. In lysed cells, metabolism was ongoing, producing metabolite changes over time, contrary to a stable metabolite content of the lysed-heated cells. These results were confirmed in both fibroblasts and adrenal cells. Therefore, in order to minimize metabolite content modifications over the measurement time, it is suggested to use cell lysis in combination with heat inactivation for extended HR-MAS NMR measurements