Human gut microbes impact host serum metabolome and insulin sensitivity.

Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus are identified as the main species driving the association between biosynthesis of BCAAs and insulin resistance, and in mice we demonstrate that P. copri can induce insulin resistance, aggravate glucose intolerance and augment circulating levels of BCAAs. Our findings suggest that microbial targets may have the potential to diminish insulin resistance and reduce the incidence of common metabolic and cardiovascular disorders.

Capillary photoionization: interface for low flow rate liquid chromatography-mass spectrometry.

This is the first report on capillary photoionization (CPI) interfacing a liquid chromatograph (LC) and mass spectrometer (MS). A new heated CPI ion source was developed, including a heated transfer capillary, a wide oval-shaped and low-depth ionization chamber with a vacuum ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to increase the photoionization efficiency and thus the sensitivity. As both analytes and eluent are first vaporized and then photoionized inside the CPI ion source between the atmosphere and the vacuum of MS, the ion transfer efficiency into the MS and thus the sensitivity is improved. The effect of the most important operation parameters, the eluent flow rate and temperature of the CPI source, on the signal intensity was studied with selected steroids. The feasibility of LC-CPI-MS/MS for the quantitative analysis of steroids was also studied in terms of linearity, repeatability, and limits of detection. The method showed good quantitative performance and sensitivity down to the low femto-mole level.

Lipidomics in biomedical research-practical considerations.

Lipids have many central physiological roles including as structural components of cell membranes, energy storage sources and intermediates in signaling pathways. Lipid-related disturbances are known to underlie many diseases and their co-morbidities. The emergence of lipidomics has empowered researchers to study lipid metabolism at the cellular as well as physiological levels at a greater depth than was previously possible. The key challenges ahead in the field of lipidomics in medical research lie in the development of experimental protocols and in silico techniques needed to study lipidomes at the systems level. Clinical questions where lipidomics may have an impact in healthcare settings also need to be identified, both from the health outcomes and health economics perspectives. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Molecular profile of the rat peri-infarct region four days after stroke: Study with MANF.

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes; however, -the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, a time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that strokeinduced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke, genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is an increase in saccharides, sugar phosphates, ceramides and free fatty acids and a decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery of AAV1-MANF to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.

Comparison of liquid chromatography-mass spectrometry and direct infusion microchip electrospray ionization mass spectrometry in global metabolomics of cell samples.

In this study, the feasibility of direct infusion electrospray ionization microchip mass spectrometry (chip-MS) was compared to the commonly used liquid chromatography-mass spectrometry (LC-MS) in non-targeted metabolomics analysis of human foreskin fibroblasts (HFF) and human induced pluripotent stem cells (hiPSC) reprogrammed from HFF. The total number of the detected features with chip-MS and LC-MS were 619 and 1959, respectively. Approximately 25% of detected features showed statistically significant changes between the cell lines with both analytical methods. The results show that chip-MS is a rapid and simple method that allows high sample throughput from small sample volumes and can detect the main metabolites and classify cells based on their metabolic profiles. However, the selectivity of chip-MS is limited compared to LC-MS and chip-MS may suffer from ion suppression.

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry.

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL-1 and limits of quantitation (LOQ) were between 0.05 to 2 ng mL-1. In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples.

Lipidome as a predictive tool in progression to type 2 diabetes in Finnish men.

BACKGROUND: There is a need for early markers to track and predict the development of type 2 diabetes mellitus (T2DM) from the state of normal glucose tolerance through prediabetes. In this study we tested whether the plasma molecular lipidome has biomarker potential to predicting the onset of T2DM. METHODS: We applied global lipidomic profiling on plasma samples from well-phenotyped men (107 cases, 216 controls) participating in the longitudinal METSIM study at baseline and at five-year follow-up. To validate the lipid markers, an additional study with a representative sample of adult male population (n=631) was also conducted. A total of 277 plasma lipids were analyzed using the lipidomics platform based on ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. Lipids with the highest predictive power for the development of T2DM were computationally selected, validated and compared to standard risk models without lipids. RESULTS: A persistent lipid signature with higher levels of triacylglycerols and diacyl-phospholipids as well as lower levels of alkylacyl phosphatidylcholines was observed in progressors to T2DM. Lysophosphatidylcholine acyl C18:2 (LysoPC(18:2)), phosphatidylcholines PC(32:1), PC(34:2e) and PC(36:1), and triacylglycerol TG(17:1/18:1/18:2) were selected to the full model that included metabolic risk factors and FINDRISC variables. When further adjusting for BMI and age, these lipids had respective odds ratios of 0.32, 2.4, 0.50, 2.2 and 0.31 (all p<0.05) for progression to T2DM. The independently-validated predictive power improved in all pairwise comparisons between the lipid model and the respective standard risk model without the lipids (integrated discrimination improvement IDI>0; p<0.05). Notably, the lipid models remained predictive of the development of T2DM in the fasting plasma glucose-matched subset of the validation study. CONCLUSION: This study indicates that a lipid signature characteristic of T2DM is present years before the diagnosis and improves prediction of progression to T2DM. Molecular lipid biomarkers were shown to have predictive power also in a high-risk group, where standard risk factors are not helpful at distinguishing progressors from non-progressors.

Molecular Atlas of Postnatal Mouse Heart Development.

Background The molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA , protein, and metabolite changes in the neonatal heart for identification of metabolism-related mechanisms associated with cardiac regeneration. Methods and Results Mouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c-means clustering were used to identify up- or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNA s and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS ( HMG -CoA [hydroxymethylglutaryl-coenzyme A] synthase)-mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU-positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle. Conclusions This study is the first systems-level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

High-grade ovarian serous carcinoma patients exhibit profound alterations in lipid metabolism.

Ovarian cancer is a very severe type of disease with poor prognosis. Treatment of ovarian cancer is challenging because of the lack of tests for early detection and effective therapeutic targets. Thus, new biomarkers are needed for both diagnostics and better understanding of the cellular processes of the disease. Small molecules, consisting of metabolites or lipids, have shown emerging potential for ovarian cancer diagnostics. Here we performed comprehensive lipidomic profiling of serum and tumor tissue samples from high-grade serous ovarian cancer patients to find lipids that were altered due to cancer and also associated with progression of the disease. Ovarian cancer patients exhibited an overall reduction of most lipid classes in their serum as compared to a control group. Despite the overall reduction, there were also specific lipids showing elevation, and especially alterations in ceramide and triacylglycerol lipid species were dependent on their fatty acyl side chain composition. Several lipids showed progressive alterations in patients with more advanced disease and poorer overall survival, and outperformed CA-125 as prognostic markers. The abundance of many serum lipids correlated with their abundance in tumor tissue samples. Furthermore, we found a negative correlation of serum lipids with 3-hydroxybutyric acid, suggesting an association between decreased lipid levels and fatty acid oxidation. In conclusion, here we present a comprehensive analysis of lipid metabolism alterations in ovarian cancer patients, with clinical implications.

Imbalance of plasma amino acids, metabolites and lipids in patients with lysinuric protein intolerance (LPI).

BACKGROUND: Lysinuric protein intolerance (LPI [MIM 222700]) is an aminoaciduria with defective transport of cationic amino acids in epithelial cells in the small intestine and proximal kidney tubules due to mutations in the SLC7A7 gene. LPI is characterized by protein malnutrition, failure to thrive and hyperammonemia. Many patients also suffer from combined hyperlipidemia and chronic kidney disease (CKD) with an unknown etiology. METHODS: Here, we studied the plasma metabolomes of the Finnish LPI patients (n=26) and healthy control individuals (n=19) using a targeted platform for analysis of amino acids as well as two analytical platforms with comprehensive coverage of molecular lipids and polar metabolites. RESULTS: Our results demonstrated that LPI patients have a dichotomy of amino acid profiles, with both decreased essential and increased non-essential amino acids. Altered levels of metabolites participating in pathways such as sugar, energy, amino acid and lipid metabolism were observed. Furthermore, of these metabolites, myo-inositol, threonic acid, 2,5-furandicarboxylic acid, galactaric acid, 4-hydroxyphenylacetic acid, indole-3-acetic acid and beta-aminoisobutyric acid associated significantly (P<0.001) with the CKD status. Lipid analysis showed reduced levels of phosphatidylcholines and elevated levels of triacylglycerols, of which long-chain triacylglycerols associated (P<0.01) with CKD. CONCLUSIONS: This study revealed an amino acid imbalance affecting the basic cellular metabolism, disturbances in plasma lipid composition suggesting hepatic steatosis and fibrosis and novel metabolites correlating with CKD in LPI. In addition, the CKD-associated metabolite profile along with increased nitrite plasma levels suggests that LPI may be characterized by increased oxidative stress and apoptosis, altered microbial metabolism in the intestine and uremic toxicity.

The influence of sample collection methodology and sample preprocessing on the blood metabolic profile.

AIM: Blood serum and plasma have intrinsic differences in their composition and the preprocessing, such as clotting temperature in serum, and storage at room temperature may have further effect on metabolite concentrations. METHODS: The influence of sampling preprocessing on the metabolic profiles in serum and different types of plasma was investigated using liquid chromatography and comprehensive 2D gas chromatography coupled to a mass spectrometer. RESULTS: The profiles of polar metabolites were significantly dependent on the type of the sample, while lipid profiles were similar in serum and different types of plasma. Extended storage of plasma at room temperature resulted in degradation of lipids already after 1 day. Serum clotting at room temperature generally resulted in higher metabolite concentration compared with serum clotting on ice.

The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes.

Colonization of the fetal and infant gut microbiome results in dynamic changes in diversity, which can impact disease susceptibility. To examine the relationship between human gut microbiome dynamics throughout infancy and type 1 diabetes (T1D), we examined a cohort of 33 infants genetically predisposed to T1D. Modeling trajectories of microbial abundances through infancy revealed a subset of microbial relationships shared across most subjects. Although strain composition of a given species was highly variable between individuals, it was stable within individuals throughout infancy. Metabolic composition and metabolic pathway abundance remained constant across time. A marked drop in alpha-diversity was observed in T1D progressors in the time window between seroconversion and T1D diagnosis, accompanied by spikes in inflammation-favoring organisms, gene functions, and serum and stool metabolites. This work identifies trends in the development of the human infant gut microbiome along with specific alterations that precede T1D onset and distinguish T1D progressors from nonprogressors.

Isoenergetic diets differing in their n-3 fatty acid and polyphenol content reflect different plasma and HDL-fraction lipidomic profiles in subjects at high cardiovascular risk.

SCOPE: Dysregulation of lipid homeostasis is related to multiple major healthcare problems. The aim of this study was to investigate the effects of n-3 fatty acid (FA) and polyphenol rich diets on plasma and HDL fraction lipidomic profiles in subjects at high cardiovascular risk. METHODS AND RESULTS: Ultra performance LC coupled to quadrupole TOF/MS mass spectrometry global lipidomic profiling was applied to plasma and HDL fraction from an 8 wk randomized intervention with four isoenergetic diets, differing in their natural n-3 FA and polyphenols content, in 78 subjects with a high BMI, abdominal obesity, and at least one other feature of the metabolic syndrome. Dependency network analysis showed a different pattern of associations between lipidomics, dietary, and clinical variables after the dietary interventions. The most remarkable associations between variables were observed after the diet high in n-3 FA and polyphenols, as the inverse association between gallic acid intake and LDL cholesterol levels, which was indirectly associated with a HDL cluster exclusively comprised lysophospholipids. CONCLUSION: This is the first human randomized controlled trial showing direct and indirect associations with lipid molecular species and clinical variables of interest in the evaluation of the metabolic syndrome after diets naturally rich in polyphenols.