Metabolomic profiling reveals mitochondrial-derived lipid biomarkers that drive obesity-associated inflammation

Obesity has reached epidemic proportions worldwide. Several animal models of obesity exist, but studies are lacking that compare traditional lard-based high fat diets (HFD) to "Cafeteria diets" (CAF) consisting of nutrient poor human junk food. Our previous work demonstrated the rapid and severe obesogenic and inflammatory consequences of CAF compared to HFD including rapid weight gain, markers of Metabolic Syndrome, multi-tissue lipid accumulation, and dramatic inflammation. To identify potential mediators of CAF-induced obesity and Metabolic Syndrome, we used metabolomic analysis to profile serum, muscle, and white adipose from rats fed CAF, HFD, or standard control diets. Principle component analysis identified elevations in clusters of fatty acids and acylcarnitines. These increases in metabolites were associated with systemic mitochondrial dysfunction that paralleled weight gain, physiologic measures of Metabolic Syndrome, and tissue inflammation in CAF-fed rats. Spearman pairwise correlations between metabolites, physiologic, and histologic findings revealed strong correlations between elevated markers of inflammation in CAF-fed animals, measured as crown like structures in adipose, and specifically the pro-inflammatory saturated fatty acids and oxidation intermediates laurate and lauroyl carnitine. Treatment of bone marrow-derived macrophages with lauroyl carnitine polarized macrophages towards the M1 pro-inflammatory phenotype through downregulation of AMPK and secretion of pro-inflammatory cytokines. Results presented herein demonstrate that compared to a traditional HFD model, the CAF diet provides a robust model for diet-induced human obesity, which models Metabolic Syndrome-related mitochondrial dysfunction in serum, muscle, and adipose, along with pro-inflammatory metabolite alterations. These data also suggest that modifying the availability or metabolism of saturated fatty acids may limit the inflammation associated with obesity leading to Metabolic Syndrome.     

Metabolomic profiling to identify potential serum biomarkers for schizophrenia and risperidone action

Despite recent advances in understanding the pathophysiology of schizophrenia and the mechanisms of antipsychotic drug action, the development of biomarkers for diagnosis and therapeutic monitoring in schizophrenia remains challenging. Metabolomics provides a powerful approach to discover diagnostic and therapeutic biomarkers by analyzing global changes in an individual's metabolic profile in response to pathophysiological stimuli or drug intervention. In this study, we performed gas chromatography-mass spectrometry based metabolomic profiling in serum of unmedicated schizophrenic patients before and after an 8-week risperidone monotherapy, to detect potential biomarkers associated with schizophrenia and risperidone treatment. Twenty-two marker metabolites contributing to the complete separation of schizophrenic patients from matched healthy controls were identified, with citrate, palmitic acid, myo-inositol, and allantoin exhibiting the best combined classification performance. Twenty marker metabolites contributing to the complete separation between posttreatment and pretreatment patients were identified, with myo-inositol, uric acid, and tryptophan showing the maximum combined classification performance. Metabolic pathways including energy metabolism, antioxidant defense systems, neurotransmitter metabolism, fatty acid biosynthesis, and phospholipid metabolism were found to be disturbed in schizophrenic patients and partially normalized following risperidone therapy. Further study of these metabolites may facilitate the development of noninvasive biomarkers and more efficient therapeutic strategies for schizophrenia.     

Metabolomic profiling of amoebic and pyogenic liver abscesses: an in vitro NMR study

Pus samples obtained from 109 patients with liver abscess were examined by NMR spectroscopy. To our knowledge this is the first report on metabolic profiling of liver abscesses. Fifty metabolites were identified by combination of one (1D) and two-dimensional (2D) NMR spectra. Metabolic derangements were evaluated for differentiation between amoebic (ALA) and pyogenic liver abscess (PLA). The NMR results indicate that aspartate, asparagine and galactose, integral components of lipoproteophophoglycans (LPG) of the cell wall of Entamoeba histolytica are metabolic biomarkers of ALA. On the other hand, acetate, propionate, butyrate, succinate and formate, the fermentation products the facultative anaerobes are significantly prevalent in PLA. The NMR based metabolic profile of ALA and PLA are evaluated taking polymerase chain reaction (PCR) and bacterial culture as gold standard method. However, when NMR results were compared with culture and PCR methods, a correct diagnosis of 94.11% in ALA (n?=?85) and 100% in PLA (n?=?10) cases were observed. NMR spectroscopy in conjunction with PCR and culture can expedite in differentiating ALA from PLA.     

Plasma phospholipid metabolic profiling and biomarkers of mouse IgA nephropathy

IgA nephropathy is the most common form of glomerulonephritis (GN) and it could progress to end-stage renal failure within 10 years. Participating in biological processes in various pathways, phospholipids as a class of important constituents in the biomembranes have been paid increasing attention in many fields. However, phospholipids metabolism in glomerular disease was not clear, especially in IgA nephropathy. In this paper, the plasma phospholipid metabolic profile in mouse IgA nephropathy was investigated to discover the potential biomarkers on the progression of this disease by using high performance liquid chromatography/mass spectrometry (HPLC/MS) and the principal components analysis (PCA) as well as partial least squares-discriminant analysis (PLS-DA). The experimental mouse models of IgA nephropathy were established by oral immune and BSA injection. It was found that expression of intercellular adhesion molecule-1 (ICAM-1) in the glomeruli had a significant correlation with proteinuria in mouse IgA nephropathy. The association between plasma phospholipids and expression of ICAM-1 in the glomeruli of IgA nephropathy suggested C18:0/C18:0 PS (phosphatidylserine), C18:0/C22:5 PS (phosphatidylserine) and C18:0/C20:4 PI (phosphatidylinositol) were possible biomarkers of IgA nephropathy. The results show that the plasma phospholipid metabolic profiles from HPLC/MS combining with PCA and PLS-DA can be used not only to differentiate the IgA nephropathy from the controls, but also to discover and identify the potential biomarkers.     

Expression profiling of liver receptor homologue 1 (LRH-1) in mouse tissues using tissue microarray

Liver receptor homologue 1 (LRH-1) is a nuclear receptor that plays important roles in lipid homeostasis and embryogenesis. To elucidate systemic physiological functions of LRH-1, we used tissue microarray-based immunohistochemistry to examine the tissue distribution and localization of LRH-1 in adult mouse tissues. LRH-1 immunoreactivity was observed in the nucleus of multiple epithelial lineage cells in the digestive system (including absorptive epithelial cells in the small and large intestines, goblet cells, acinar cells of the exocrine glands, chief cells and mucus neck cells in the stomach, granular and prickle layer cells in the tongue and forestomach, and gall bladder epithelium); respiratory system (alveolar type II cells); and urinary system (transitional epithelium). Nuclear LRH-1 immunoreactivity was also localized in cells involved in fatty acid/glucose metabolism, including hepatocytes, brown adipocytes, and cardiomyocytes, and neurons involved in the regulation of food intake, including the arcuate nucleus in the hypothalamus and paraventricular nucleus of thalamus. Additionally, LRH-1 immunoreactivity was observed in testicular Leydig cells and ovarian follicular cells. These data suggest that LRH-1 functions in multiple organ systems to regulate epithelial cell physiology and differentiation, energy metabolism, and reproduction. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metabolomic evaluation of rat liver and testis to characterize the toxicity of triazole fungicides

The effects of two triazole fungicides, myclobutanil and triadimefon, on endogenous rat metabolite profiles in blood serum, liver, and testis was assessed using proton nuclear magnetic resonance (¹H-NMR) spectroscopy. Adult male Sprague-Dawley rats were dosed daily by gavage for 14 days with myclobutanil or triadimefon, at two dose levels for each triazole. Following exposure, serum, liver, and testis were collected and processed for NMR analysis. Principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the resulting spectra were used to determine changes in metabolite profiles as a result of exposure. Using this approach, responses common to both triazoles were identified, as well as responses indicative of differences in the toxicity of these two compounds. Although changes were observed in serum metabolites following exposure, none were robust enough to be considered a biomarker of exposure/effect. A number of metabolic changes were, however, observed in the liver with both triazoles, particularly in metabolites related to the methionine cycle. The testes of myclobutanil-exposed animals displayed altered levels of creatine and creatinine, consistent with testicular toxicity. Overall, the results of this study support the possible application of a metabolomics approach to assessing the toxicity of triazole fungicides and identifying biomarkers of exposure and/or effect.RNM supported by EPA/NCSU Cooperative Training Agreement #CT826512010 with the Department of Environmental and Molecular Toxicology, North Caroline State University, Raleigh.The research described in this article has been reviewed by the U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and the policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.     

Transcriptional profiling of mouse liver in response to chronic heat stress

Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 °C) or control (24 °C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.     

Metabolomic profiling and antioxidant activity of some Acacia species

Metabolomic profiling of different parts (leaves, flowers and pods) of Acacia species (Acacia nilotica, Acacia seyal and Acacia laeta) was evaluated. The multivariate data analyses such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were used to differentiate the distribution of plant metabolites among different species or different organs of the same species. A.nilotica was characterized with a high content of saponins and A.seyal was characterized with high contents of proteins, phenolics, flavonoids and anthocyanins. A.laeta had a higher content of carbohydrates than A. nilotica and A. seyal. On the basis of these results, total antioxidant capacity, DPPH free radical scavenging activity and reducing power of the methanolic extracts of studied parts were evaluated. A.nilotica and A.seyal extracts showed less inhibitory concentration 50 (IC₅₀) compared to A.laeta extracts which means that these two species have the strongest radical scavenging activity whereas A. laeta extracts have the lowest radical scavenging activity. A positive correlation between saponins and flavonoids with total antioxidant capacity and DPPH radical scavenging activity was observed. Based on these results, the potentiality of these plants as antioxidants was discussed.     

Metabolomic profiling in follicular fluid of patients with infertility-related deep endometriosis

Introduction

Endometriosis is an estrogen-dependent gynecological disease that causes infertility, and potential metabolomic biomarkers related to ovarian endometriosis and poor outcomes after assisted reproductive treatments are still lacking.

Objectives

The present study analyzed the metabolomic profiling of follicular fluid samples from 40 patients undergoing in vitro fertilization.

Methods

The follicular fluid samples were classified as controls (n = 22) and endometriosis patients (n = 18). The samples were submitted to Bligh and Dyer protocol followed by metabolomics analysis by ultra-performance liquid chromatography mass spectrometry. Clinical data was assessed by Students’ T-test and metabolomics data was analyzed by multivariate statistics by MetaboAnalyst 3.0 to obtain intrinsic characteristics that allowed for groups discrimination. The Receiver Operating Characteristic curve was carried out for the proposed biomarkers, aiming to determine their specificity and sensitivity, as a set and individually.

Results

From the metabolomic analysis, 20 ion masses were selected as potential biomarkers from principal component analysis, which showed that all biomarkers were more abundant in the endometriosis group when compared to controls. Tentative attribution was performed by lipid maps database, demonstrating that these potential biomarkers correspond to fatty acids, carnitines, monoacylglycerols, lysophosphatidic acids, lysophosphatidylglycerols, diacylglycerols, lysophosphatidylcholines, phosphatidylserine, lysophosphatidylinositols and Phosphatidic Acid.

Conclusion

The use of mass spectrometry-based metabolomics allowed for the identification of effective biomarkers for ovarian endometriosis, which may contribute for a better comprehension of the disease and how it affects the ovary, as well as assisting in the development of accessory tools for endometriosis diagnosis and infertility management.

     

Metabolomic profiling of the effects of allopurinol on Drosophila melanogaster

Metabolomic profiling using hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry was used to study the effects of the xanthine oxidase inhibitor allopurinol on wild type Drosophila melanogaster. Allopurinol treatment phenocopied the rosy mutation causing an elevation in the levels of xanthine and hypoxanthine and a fall in the levels of uric acid and allantoin. However, in addition there were some unexpected metabolic changes after treatment. Ascorbic acid levels were undetectable, glutathione levels fell and glutathione disulphide levels rose, methionine S-oxide levels rose and riboflavin levels fell. The origin of this oxidative stress was not immediately apparent; however, there was a strong suggestion that it might be related to a fall in NADPH levels linked to a reduction in glucose-6-phosphate dehydrogenase activity, resulting in reduced levels of some metabolites in the pentose phosphate pathway. In addition to producing oxidative stress there were marked effects on tryptophan metabolism with most of the metabolites in the kynurenine pathway being lowered by allopurinol treatment. The effects on the kynurenine pathway could be related to the established use of allopurinol in treating schizophrenia.     

Metabolomic profiling from formalin-fixed, paraffin-embedded tumor tissue using targeted LC/MS/MS: application in sarcoma

The relatively new field of onco-metabolomics attempts to identify relationships between various cancer phenotypes and global metabolite content. Previous metabolomics studies utilized either nuclear magnetic resonance spectroscopy or gas chromatography/mass spectrometry, and analyzed metabolites present in urine and serum. However, direct metabolomic assessment of tumor tissues is important for determining altered metabolism in cancers. In this respect, the ability to obtain reliable data from archival specimens is desirable and has not been reported to date. In this feasibility study, we demonstrate the analysis of polar metabolites extracted directly from ten formalin-fixed, paraffin-embedded (FFPE) specimens, including five soft tissue sarcomas and five paired normal samples. Using targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) via selected reaction monitoring (SRM), we detect an average of 106 metabolites across the samples with excellent reproducibility and correlation between different sections of the same specimen. Unsupervised hierarchical clustering and principal components analysis reliably recovers a priori known tumor and normal tissue phenotypes, and supervised analysis identifies candidate metabolic markers supported by the literature. In addition, we find that diverse biochemical processes are well-represented in the list of detected metabolites. Our study supports the notion that reliable and broadly informative metabolomic data may be acquired from FFPE soft tissue sarcoma specimens, a finding that is likely to be extended to other malignancies.     

Metabolomic identification of diagnostic serum-based biomarkers for advanced stage melanoma

Introduction

Melanoma is a highly aggressive malignancy and is currently one of the fastest growing cancers worldwide. While early stage (I and II) disease is highly curable with excellent prognosis, mortality rates rise dramatically after distant spread. We sought to identify differences in the metabolome of melanoma patients to further elucidate the pathophysiology of melanoma and identify potential biomarkers to aid in earlier detection of recurrence.

Methods

Using ¹H NMR and DI–LC–MS/MS, we profiled serum samples from 26 patients with stage III (nodal metastasis) or stage IV (distant metastasis) melanoma and compared their biochemical profiles with 46 age- and gender-matched controls.

Results

We accurately quantified 181 metabolites in serum using a combination of ¹H NMR and DI–LC–MS/MS. We observed significant separation between cases and controls in the PLS-DA scores plot (permutation test p-value?=?0.002). Using the concentrations of PC-aa-C40:3, dl-carnitine, octanoyl-l-carnitine, ethanol, and methylmalonyl-l-carnitine we developed a diagnostic algorithm with an AUC (95% CI)?=?0.822 (0.665–0.979) with sensitivity and specificity of 100 and 56%, respectively. Furthermore, we identified arginine, proline, tryptophan, glutamine, glutamate, glutathione and ornithine metabolism to be significantly perturbed due to disease (p?<?0.05).

Conclusion

Targeted metabolomic analysis demonstrated significant differences in metabolic profiles of advanced stage (III and IV) melanoma patients as compared to controls. These differences may represent a potential avenue for the development of multi-marker serum-based assays for earlier detection of recurrences, allow for newer, more effective targeted therapy when tumor burden is less, and further elucidate the pathophysiologic changes that occur in melanoma.

     

Metabolomic profiling of rapid cold hardening and cold shock in Drosophila melanogaster

A short exposure to a mild cold stress is sufficient to increase cold tolerance in many insects. This phenomenon, termed rapid cold hardening (RCH) expands the thermal interval that can be exploited by the insect. To investigate the possible role of altered metabolite levels during RCH, the present study used untargeted (1)H NMR metabolomic profiling to examine the metabolomic response in Drosophila melanogaster during the 72 h following RCH and cold shock treatment. These findings are discussed in relation to the costs and benefits of RCH that are measured in terms of survival and reproductive output. Cold shock caused a persistent disturbance of the metabolite profile that correlated well with a delayed onset of cold shock mortality. The disruption of metabolite homeostasis was smaller following RCH, where control levels were fully recovered after 72 h. RCH improved both survival and reproductive output after a subsequent cold shock but the RCH treatment alone was associated with costs in terms of reduced survival and reproductive output. The most pronounced changes following the RCH treatment were elevated levels of glucose and trehalose. Although, it is difficult to discern if a change in a specific metabolite is linked to physiological processes of adaptive, neutral or detrimental nature we observed that the onset and magnitude of the increased sugar levels correlated tightly with the improved chill tolerance following RCH. These findings suggest a putative role of cryoprotectants during RCH which are discussed in the light of the existing literature on the mechanistic background of RCH.