Elevated levels of branched-chain amino acids (BCAAs) have recently been implicated in the development of cardiovascular and metabolic diseases, but the molecular mechanisms are unknown. In a mouse ...model of impaired BCAA catabolism (knockout KO), we found that chronic accumulation of BCAAs suppressed glucose metabolism and sensitized the heart to ischemic injury. High levels of BCAAs selectively disrupted mitochondrial pyruvate utilization through inhibition of pyruvate dehydrogenase complex (PDH) activity. Furthermore, downregulation of the hexosamine biosynthetic pathway in KO hearts decreased protein O-linked N-acetylglucosamine (O-GlcNAc) modification and inactivated PDH, resulting in significant decreases in glucose oxidation. Although the metabolic remodeling in KO did not affect baseline cardiac energetics or function, it rendered the heart vulnerable to ischemia-reperfusion injury. Promoting BCAA catabolism or normalizing glucose utilization by overexpressing GLUT1 in the KO heart rescued the metabolic and functional outcome. These observations revealed a novel role of BCAA catabolism in regulating cardiac metabolism and stress response.
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•Branched-chain amino acid (BCAA) catabolism regulates glucose metabolism in the heart•High levels of BCAAs selectively disrupt mitochondrial pyruvate utilization•Downregulation of HBP by chronic accumulation of BCAAs inactivates PDH•Defective BCAA catabolism sensitizes the heart to ischemia-reperfusion insult
Branched-chain amino acids (BCAAs) have been implicated in cardiovascular disease. Li et al. now reveal molecular mechanisms behind BCAA catabolism in regulating cardiac metabolism and stress response. Chronic accumulation of BCAAs downregulates the hexosamine biosynthetic pathway and inactivates pyruvate dehydrogenase, which renders the heart vulnerable to ischemic injury.
Quantitative NMR-based metabolite profiling is challenged by the deleterious effects of abundant proteins in the intact blood plasma/serum, which underscores the need for alternative approaches. ...Protein removal by ultrafiltration using low molecular weight cutoff filters thus represents an important step. However, protein precipitation, an alternative and simple approach for protein removal, lacks detailed quantitative assessment for use in NMR based metabolomics. In this study, we have comprehensively evaluated the performance of protein precipitation using methanol, acetonitrile, perchloric acid, and trichloroacetic acid and ultrafiltration approaches using 1D and 2D NMR, based on the identification and absolute quantitation of 44 human blood metabolites, including a few identified for the first time in the NMR spectra of human serum. We also investigated the use of a “smart isotope tag,” 15N-cholamine for further resolution enhancement, which resulted in the detection of a number of additional metabolites. 1H NMR of both protein precipitated and ultrafiltered serum detected all 44 metabolites with comparable reproducibility (average CV, 3.7% for precipitation; 3.6% for filtration). However, nearly half of the quantified metabolites in ultrafiltered serum exhibited 10–74% lower concentrations; specifically, tryptophan, benzoate, and 2-oxoisocaproate showed much lower concentrations compared to protein precipitated serum. These results indicate that protein precipitation using methanol offers a reliable approach for routine NMR-based metabolomics of human blood serum/plasma and should be considered as an alternative to ultrafiltration. Importantly, protein precipitation, which is commonly used by mass spectrometry (MS), promises avenues for direct comparison and correlation of metabolite data obtained from the two analytical platforms to exploit their combined strength in the metabolomics of blood.
Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the ...pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics.
The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes.
We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload-induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy.
Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.
A current challenge in metabolomics is the reliable quantitation of many metabolites. Limited resolution and sensitivity combined with the challenges associated with unknown metabolite identification ...have restricted both the number and the quantitative accuracy of blood metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database searches, and spiking with authentic compounds enabled the identification of 67 blood metabolites. Many of these (∼1/3) are new compared with those reported previously as a part of the Human Serum Metabolome Database. In addition, considering both the high reproducibility and quantitative nature of NMR as well as the sensitivity of NMR chemical shifts to altered sample conditions, experimental protocols and comprehensive peak annotations are provided here as a guide for identification and quantitation of the new pool of blood metabolites for routine applications. Further, investigations focused on the evaluation of quantitation using organic solvents revealed a surprisingly poor performance for protein precipitation using acetonitrile. One-third of the detected metabolites were attenuated by 10–67% compared with methanol precipitation at the same solvent-to-serum ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1 (v/v). These results, combined with the newly established identity for many unknown metabolites in the NMR spectrum, offer new avenues for human serum/plasma-based metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that represent numerous classes, including amino acids, organic acids, carbohydrates, and heterocyclic compounds, using a simple and highly reproducible analytical method such as NMR may potentially guide the evaluation of samples for analysis using mass spectrometry.
Over the past two decades, nuclear magnetic resonance (NMR) has emerged as one of the three principal analytical techniques used in metabolomics (the other two being gas chromatography coupled to ...mass spectrometry (GC-MS) and liquid chromatography coupled with single-stage mass spectrometry (LC-MS)). The relative ease of sample preparation, the ability to quantify metabolite levels, the high level of experimental reproducibility, and the inherently nondestructive nature of NMR spectroscopy have made it the preferred platform for long-term or large-scale clinical metabolomic studies. These advantages, however, are often outweighed by the fact that most other analytical techniques, including both LC-MS and GC-MS, are inherently more sensitive than NMR, with lower limits of detection typically being 10 to 100 times better. This review is intended to introduce readers to the field of NMR-based metabolomics and to highlight both the advantages and disadvantages of NMR spectroscopy for metabolomic studies. It will also explore some of the unique strengths of NMR-based metabolomics, particularly with regard to isotope selection/detection, mixture deconvolution via 2D spectroscopy, automation, and the ability to noninvasively analyze native tissue specimens. Finally, this review will highlight a number of emerging NMR techniques and technologies that are being used to strengthen its utility and overcome its inherent limitations in metabolomic applications.
In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite ...succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.
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•Expression of receptors enabling chemosensing on tuft cells is tissue specific•Tuft cells in the small intestine express the succinate receptor SUCNR1•Succinate is sufficient to induce a multifaceted type 2 immune response•Immune sensing of Tritrichomonas colonization by tuft cells requires SUCNR1
Tuft cells have been proposed to act as immune sentinels in multiple tissues. Nadjsombati and McGinty et al. now show that detection of the microbial metabolite succinate by tuft cells in the small intestine is sufficient to induce a type 2 immune response, suggesting that tuft cells monitor microbial metabolites to initiate type 2 immunity.
Glucose and branched-chain amino acids (BCAAs) are essential nutrients and key determinants of cell growth and stress responses. High BCAA level inhibits glucose metabolism but reciprocal regulation ...of BCAA metabolism by glucose has not been demonstrated. Here we show that glucose suppresses BCAA catabolism in cardiomyocytes to promote hypertrophic response. High glucose inhibits CREB stimulated KLF15 transcription resulting in downregulation of enzymes in the BCAA catabolism pathway. Accumulation of BCAA through the glucose-KLF15-BCAA degradation axis is required for the activation of mTOR signaling during the hypertrophic growth of cardiomyocytes. Restoration of KLF15 prevents cardiac hypertrophy in response to pressure overload in wildtype mice but not in mutant mice deficient of BCAA degradation gene. Thus, regulation of KLF15 transcription by glucose is critical for the glucose-BCAA circuit which controls a cascade of obligatory metabolic responses previously unrecognized for cell growth.
Colorectal cancer (CRC) is one of the most prevalent and deadly cancers in the world. Despite an expanding knowledge of its molecular pathogenesis during the past two decades, robust biomarkers to ...enable screening, surveillance, and therapy monitoring of CRC are still lacking. In this study, we present a targeted liquid chromatography–tandem mass spectrometry-based metabolic profiling approach for identifying biomarker candidates that could enable highly sensitive and specific CRC detection using human serum samples. In this targeted approach, 158 metabolites from 25 metabolic pathways of potential significance were monitored in 234 serum samples from three groups of patients (66 CRC patients, 76 polyp patients, and 92 healthy controls). Partial least-squares–discriminant analysis (PLS–DA) models were established, which proved to be powerful for distinguishing CRC patients from both healthy controls and polyp patients. Receiver operating characteristic curves generated based on these PLS–DA models showed high sensitivities (0.96 and 0.89, respectively, for differentiating CRC patients from healthy controls or polyp patients), good specificities (0.80 and 0.88), and excellent areas under the curve (0.93 and 0.95). Monte Carlo cross validation was also applied, demonstrating the robust diagnostic power of this metabolic profiling approach.
Breast cancer detection using targeted plasma metabolomics Jasbi, Paniz; Wang, Dongfang; Cheng, Sunny Lihua ...
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences,
01/2019, Letnik:
1105
Journal Article
Recenzirano
Breast cancer (BC) is a major cause of human morbidity and mortality, especially among women. Despite the important role of metabolism in the molecular pathogenesis of cancer, robust metabolic ...markers to enable enhanced screening and disease monitoring of BC are still critically needed. In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolic profiling approach is presented for the identification of metabolic marker candidates that could enable highly sensitive and specific detection of all-stage as well as early-stage BC. In this targeted approach, 105 metabolites from >35 metabolic pathways of potential biological relevance were reliably detected in 201 plasma samples taken from two groups of subjects (102 BC patients and 99 healthy controls). The results of our general linear model and partial least squares-discriminant analysis (PLS-DA) informed the construction of a novel 6-metabolite panel of potential biomarkers. A receiver operating characteristic (ROC) curve generated based on an improved PLS-DA model showed relatively high sensitivity (0.80), specificity (0.75), and area under the receiver-operating characteristic curve (AUROC = 0.89). Similar classification performance of the model was observed for detection of early-stage BC (AUROC = 0.87, sensitivity: 0.86, specificity: 0.75). Bioinformatics analyses revealed significant disturbances in arginine/proline metabolism, tryptophan metabolism, and fatty acid biosynthesis. Our univariate and multivariate results indicate the effectiveness of this metabolomics approach for all-stage as well as early-stage BC diagnosis; our bioinformatics results indicate affected pathways related to tumor growth, metastasis, and immune escape mechanisms. Future studies should validate these results using more samples from different locations.
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