Cross-comparison of systemic and tissue-specific metabolomes in a mouse model of Leigh syndrome.

INTRODUCTION: The value of metabolomics in multi-systemic mitochondrial disease research has been increasingly recognized, with the ability to investigate a variety of biofluids and tissues considered a particular advantage. Although minimally invasive biofluids are the generally favored sample type, it remains unknown whether systemic metabolomes provide a clear reflection of tissue-specific metabolic alterations. OBJECTIVES: Here we cross-compare urine and tissue-specific metabolomes in the Ndufs4 knockout mouse model of Leigh syndrome-a complex neurometabolic MD defined by progressive focal lesions in specific brain regions-to identify and evaluate the extent of common and unique metabolic alterations on a systemic and brain regional level. METHODS: Untargeted and semi-targeted multi-platform metabolomics were performed on urine, four brain regions, and two muscle types of Ndufs4 KO (n≥19) vs wildtype (n≥20) mice. RESULTS: Widespread alterations were evident in alanine, aspartate, glutamate, and arginine metabolism in Ndufs4 KO mice; while brain-region specific metabolic signatures include the accumulation of branched-chain amino acids, proline, and glycolytic intermediates. Furthermore, we describe a systemic dysregulation in one-carbon metabolism and the tricarboxylic acid cycle, which was not clearly reflected in the Ndufs4 KO brain. CONCLUSION: Our results confirm the value of urinary metabolomics when evaluating MD-associated metabolites, while cautioning against mechanistic studies relying solely on systemic biofluids.

Sub-Cellular Metabolomics Contributes Mitochondria-Specific Metabolic Insights to a Mouse Model of Leigh Syndrome.

Direct injury of mitochondrial respiratory chain (RC) complex I by Ndufs4 subunit mutations results in complex I deficiency (CID) and a progressive encephalomyopathy, known as Leigh syndrome. While mitochondrial, cytosolic and multi-organelle pathways are known to be involved in the neuromuscular LS pathogenesis, compartment-specific metabolomics has, to date, not been applied to murine models of CID. We thus hypothesized that sub-cellular metabolomics would be able to contribute organelle-specific insights to known Ndufs4 metabolic perturbations. To that end, whole brains and skeletal muscle from late-stage Ndufs4 mice and age/sex-matched controls were harvested for mitochondrial and cytosolic isolation. Untargeted 1H-NMR and semi-targeted LC-MS/MS metabolomics was applied to the resulting cell fractions, whereafter important variables (VIPs) were selected by univariate statistics. A predominant increase in multiple targeted amino acids was observed in whole-brain samples, with a more prominent effect at the mitochondrial level. Similar pathways were implicated in the muscle tissue, showing a greater depletion of core metabolites with a compartment-specific distribution, however. The altered metabolites expectedly implicate altered redox homeostasis, alternate RC fueling, one-carbon metabolism, urea cycling and dysregulated proteostasis to different degrees in the analyzed tissues. A first application of EDTA-chelated magnesium and calcium measurement by NMR also revealed tissue- and compartment-specific alterations, implicating stress response-related calcium redistribution between neural cell compartments, as well as whole-cell muscle magnesium depletion. Altogether, these results confirm the ability of compartment-specific metabolomics to capture known alterations related to Ndufs4 KO and CID while proving its worth in elucidating metabolic compartmentalization in said pathways that went undetected in the diluted whole-cell samples previously studied.

Time-based investigation of urinary metabolic markers for Type 2 diabetes: Metabolomics approach for diabetes management.

Diabetes is considered one of the most important health emergencies worldwide and Egypt has 8.2 million diabetic patients according to the International Diabetes Federation report in 2017. The objective of this study was to monitor the time-course variation in the metabolic profile of diabetic rats to detect urinary metabolic biomarkers using the metabolomics approach. Type 2 diabetes was induced in male Wistar albino rats using a single intraperitoneal injection of 40 mg/kg of streptozotocin following oral administration of 10% fructose in drinking water for 3 weeks. Then, urine was collected for 24 h from rats at three time points (0, 2, and 4 weeks after confirmation of diabetes), and were analyzed by nuclear magnetic resonance (H1 -NMR), followed by multivariate data analysis. The results from H1 -NMR pointed out that d-glucose, taurine, l-carnitine, l-fucose, 1,5-anhydrosorbitol, and d-galactose levels showed consistent significant variation (p < 0.05) between the positive (diabetic) and negative (normal) controls during the whole experimental period. Also, with the disease progression, myoinositol, and l-phenylalanine levels were significantly altered (p < 0.05) after 2 weeks and this alteration was maintained till the end of the 4-week experimental period in the positive control group. From the results of the present study, it could be concluded that we cannot depend only on glucose levels for prognostic purposes since there are other metabolic disturbances in diabetes which need to be tracked for better disease prognosis.

The cross-tissue metabolic response of abalone (Haliotis midae) to functional hypoxia.

Functional hypoxia is a stress condition caused by the abalone itself as a result of increased muscle activity, which generally necessitates the employment of anaerobic metabolism if the activity is sustained for prolonged periods. With that being said, abalone are highly reliant on anaerobic metabolism to provide partial compensation for energy production during oxygen-deprived episodes. However, current knowledge on the holistic metabolic response for energy metabolism during functional hypoxia, and the contribution of different metabolic pathways and various abalone tissues towards the overall accumulation of anaerobic end-products in abalone are scarce. Metabolomics analysis of adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue samples indicated that South African abalone (Haliotis midae) subjected to functional hypoxia utilises predominantly anaerobic metabolism, and depends on all of the main metabolite classes (proteins, carbohydrates and lipids) for energy supply. Functional hypoxia caused increased levels of anaerobic end-products: lactate, alanopine, tauropine, succinate and alanine. Also, elevation in arginine levels was detected, confirming that abalone use phosphoarginine to generate energy during functional hypoxia. Different tissues showed varied metabolic responses to hypoxia, with functional hypoxia showing excessive changes in the adductor muscle and gills. From this metabolomics investigation, it becomes evident that abalone are metabolically able to produce sufficient amounts of energy when functional hypoxia is experienced. Also, tissue interplay enables the adjustment of H. midae energy requirements as their metabolism shifts from aerobic to anaerobic respiration during functional hypoxia.This article has an associated First Person interview with the first author of the paper.

The effect of temperature on the respiration and metabolism of the African burrowing scorpion (Opistophthalmus latimanus).

It is well known that scorpions are highly adapted to thermal temperatures. However, little is known of the metabolic and respiration adaptations caused by temperature fluctuations in these animals. Therefore we used the African burrowing scorpion Opistophthalmus latimanus to measure the effect of temperature on its metabolism and respiration. Radioactive d-glucose was injected into the ventral sinus of the circulatory system and metabolites of d-glucose were determined after six hour incubation at four temperatures (7, 17, 25 and 37°C). The oxygen consumption rate (MO2) and carbon dioxide production rate (MCO2) were measured simultaneously at 17, 25 and 37°C. The metabolomics investigation included LC-MS, GC-MS and NMR analytical platforms. The average radioactivity recovered after the carbon-14 d-glucose injection, glycogen precipitation and column fractionation at the four temperatures was between 92.4% and 95.0%. Strong acids, CO2 and neutral compounds all increased with temperature, while glycogen and neutral sugars decreased as the temperature increased. Weak acids initially increased with temperature, then decreased again as the temperature was increased to 37°C. Respiration also gradually increased as the temperature was increased. Metabolomics identified 23 metabolites that were significantly influenced by temperature. Pathway analysis of these metabolites indicated numerous metabolic pathways that were affected by temperature, clearly demonstrating that the scorpion uses proteins, lipids and carbohydrates at higher temperatures to generate energy. However, protein catabolism seems to be the main source of energy at higher temperatures in these animals, although this needs to be confirmed in a more targeted metabolomics study.

Obesity and metabolomics: metallothioneins protect against high-fat diet-induced consequences in metallothionein knockout mice.

Obesity continues to rise as an alarming global epidemic. System level mechanisms, diagnostics, and therapeutics are sorely needed so as to identify at risk individuals and design appropriate population scale interventions. The present study evaluated the protective role of metallothioneins (MTs) against obesity and high-fat diet-induced effects such as insulin resistance in both male and female MT-1+2 knockout and MT-3 knockout mice. As the metabolome is closest to the functional phenotype, changes in metabolite levels were also evaluated, and the direct or indirect involvement of MTs in metabolism examined. MT-1+2-, MT-3 knockout, and wild-type mice were given a high-fat diet for 2 months. Variation in body weight gain, tissue weight, and response to oral glucose tolerance test and insulin tolerance test were determined and compared to mice that received the control diet. Effect of the high-fat diet on the knockout mice were investigated on the metabolome level in specific tissues using metabolomics. Both knockout mice strains were more susceptible to high-fat diet-induced effects, such as weight gain and moderate insulin resistance, with the MT-3 knockout mice most susceptible. Brain tissue of the knockout mice showed most metabolic variation and pointed to possible impairment of mitochondrial function. The protective effect of MTs against high-fat diet and obesity-induced effects such as insulin resistance was evident from our observations. The putative role MTs play in mitochondrial function is possibly the main contributor to the lack of these effects in wild-type mice. Considering the expression profiles of the MT isoforms and similarity in brain metabolic variation in the knockout strains, it appears that they promote mitochondrial function in the hypothalamus, thereby limiting weight gain and insulin resistance. Furthermore, metabolomics research in preclinical models of obesity and in the clinic is warranted in the near future.

Use of metabolomics to elucidate the metabolic perturbation associated with hypertension in a black South African male cohort: the SABPA study.

There is concern about the increasing burden of essential hypertension in urban-dwelling black South Africans, especially males. Several studies have investigated urbanization and hypertension in South Africans, but in-depth metabolomics studies on these urbanized hypertensives are still lacking. We aimed to investigate hypertension via two metabolomics methods in order to explore underlying biological mechanisms, demonstrating the effectiveness of these methods in cardiovascular research. A comprehensive characterization of a group (n = 25) of black male South Africans was performed using urinary gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry metabolic profiling in conjunction with 24-hour ambulatory blood pressure readings and anthropometric, clinical, and biochemical markers. Average 24-hour blood pressure readings served as the grouping variable, and test subjects were divided into quintiles. Statistical analyses were performed on Quintile 1 (normotensive subjects) and Quintile 5 (extreme hypertensive subjects). After feature selection was performed, several metabolites and cardiometabolic risk markers, including abdominal obesity and markers of liver damage, inflammation, and oxidative stress were significantly perturbed in Quintile 5 (hypertensives) compared with Quintile 1 (P < .05). Pathway analysis revealed perturbations in several systems involved in ethanol metabolism via shifted global NADH/NAD(+) ratio. Although alcohol abuse has been established as a risk factor for hypertension, this study illustrated a metabolic perturbation associated with alcohol abuse, contributing to the development of hypertension-possibly by altering bioenergetics through a shift in the NADH/NAD(+) ratio. Following this finding, future intervention studies on alcohol moderation, as well as further enhancement of metabolomics methods in cardiovascular research are highly recommended.