Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology.

Herbal remedies are increasing in popularity as treatments for metabolic conditions such as obesity and Type 2 Diabetes. One potential therapeutic option is fenugreek seeds (Trigonella foenum-graecum), which have been used for treating high cholesterol and Type 2 diabetes. A proposed mechanism for these benefits is through alterations in the microbiome, which impact mammalian host metabolic function. This study used untargeted metabolomics to investigate the fenugreek-induced alterations in the intestinal, liver, and serum profiles of mice fed either a 60% high-fat or low-fat control diet each with or without fenugreek supplementation (2% w/w) for 14 weeks. Metagenomic analyses of intestinal contents found significant alterations in the relative composition of the gut microbiome resulting from fenugreek supplementation. Specifically, Verrucomicrobia, a phylum containing beneficial bacteria which are correlated with health benefits, increased in relative abundance with fenugreek. Metabolomics partial least squares discriminant analysis revealed substantial fenugreek-induced changes in the large intestines. However, it was observed that while the magnitude of changes was less, significant modifications were present in the liver tissues resulting from fenugreek supplementation. Further analyses revealed metabolic processes affected by fenugreek and showed broad ranging impacts in multiple pathways, including carnitine biosynthesis, cholesterol and bile acid metabolism, and arginine biosynthesis. These pathways may play important roles in the beneficial effects of fenugreek.

Safety and pharmacokinetics of naringenin: A randomized, controlled, single-ascending-dose clinical trial.

AIMS: To evaluate the safety and pharmacokinetics of naringenin in healthy adults consuming whole-orange (Citrus sinensis) extract. METHODS AND METHODS: In a single-ascending-dose randomized crossover trial, 18 adults ingested doses of 150 mg (NAR150), 300 mg (NAR300), 600 mg (NAR600) and 900 mg (NAR900) naringenin or placebo. Each dose or placebo was followed by a wash-out period of at least 1 week. Blood safety markers were evaluated pre-dose and 24 hours post-dose. Adverse events (AEs) were recorded. Serum naringenin concentrations were measured before and over 24 hours following ingestion of placebo, NAR150 and NAR600. Four- and 24-hour serum measurements were obtained after placebo, NAR300 and NAR900 ingestion. Data were analysed using a mixed-effects linear model. RESULTS: There were no relevant AEs or changes in blood safety markers following ingestion of any of the naringenin doses. The pharmacokinetic variables were: maximal concentration: 15.76 ± 7.88 µM (NAR150) and 48.45 ± 7.88 µM (NAR600); time to peak: 3.17 ± 0.74 hours (NAR150) and 2.41 ± 0.74 hours (NAR600); area under the 24-hour concentration-time curve: 67.61 ± 24.36 µM × h (NAR150) and 199.05 ± 24.36 µM × h (NAR600); and apparent oral clearance: 10.21 ± 2.34 L/h (NAR150) and 13.70 ± 2.34 L/h (NAR600). Naringenin half-life was 3.0 hours (NAR150) and 2.65 hours (NAR600). After NAR300 ingestion, serum concentrations were 10.67 ± 5.74 µM (4 hours) and 0.35 ± 0.30 µM (24 hours). After NAR900 ingestion, serum concentrations were 43.11 ± 5.26 µM (4 hours) and 0.24 ± 0.30 µM (24 hours). CONCLUSIONS: Ingestion of 150 to 900 mg doses of naringenin is safe in healthy adults, and serum concentrations are proportional to the dose administered. Since naringenin (8 µM) is effective in primary human adipocytes, ingestion of 300 mg naringenin twice/d will likely elicit a physiological effect.

Expanding lipidomics coverage: effective ultra performance liquid chromatography-high resolution mass spectrometer methods for detection and quantitation of cardiolipin, phosphatidylglycerol, and lysyl-phosphatidylglycerol.

INTRODUCTION: Lipidomics can reveal global alterations in a broad class of molecules whose functions are innately linked to physiology. Monitoring changes in the phospholipid composition of biological membranes in response to stressors can aid the development of targeted therapies. However, exact quantitation of cardiolipins is not a straightforward task due to low ionization efficiencies and poor chromatographic separation of these compounds. OBJECTIVE: The aim of this study was to develop a quantitative method for the detection of cardiolipins and other phospholipids using both a targeted and untargeted analyses with a Q-Exactive. METHODS: HILIC chromatography and high-resolution mass spectrometry with parallel reaction monitoring was used to measure changes in lipid concentration. Internal standards and fragmentation techniques allowed for the reliable quantitation of lipid species including: lysyl-phosphatidylglycerol, phosphatidylglycerol, and cardiolipin. RESULTS: The untargeted analysis was capable to detecting 6 different phospholipid classes as well as free fatty acids. The targeted analysis quantified up to 23 cardiolipins, 10 phosphatidylglycerols and 10 lysyl-phosphatidylglycerols with detection limits as low as 50 nM. Biological validation with Enterococcus faecalis demonstrates sensitivity in monitoring the incorporation of exogenously supplied free fats into membrane phospholipids. When supplemented with oleic acid, the amount of free oleic acid in the membrane was 100 times greater and the concentration of polyunsaturated cardiolipin increased to over 3.5 µM compared to controls. CONCLUSIONS: This lipidomics method is capable of targeted quantitation for challenging biologically relevant cardiolipins as well as broad, untargeted lipid profiling.

Metabolomic analysis of mouse prefrontal cortex reveals upregulated analytes during wakefulness compared to sleep.

By identifying endogenous molecules in brain extracellular fluid metabolomics can provide insight into the regulatory mechanisms and functions of sleep. Here we studied how the cortical metabolome changes during sleep, sleep deprivation and spontaneous wakefulness. Mice were implanted with electrodes for chronic sleep/wake recording and with microdialysis probes targeting prefrontal and primary motor cortex. Metabolites were measured using ultra performance liquid chromatography-high resolution mass spectrometry. Sleep/wake changes in metabolites were evaluated using partial least squares discriminant analysis, linear mixed effects model analysis of variance, and machine-learning algorithms. More than 30 known metabolites were reliably detected in most samples. When used by a logistic regression classifier, the profile of these metabolites across sleep, spontaneous wake, and enforced wake was sufficient to assign mice to their correct experimental group (pair-wise) in 80-100% of cases. Eleven of these metabolites showed significantly higher levels in awake than in sleeping mice. Some changes extend previous findings (glutamate, homovanillic acid, lactate, pyruvate, tryptophan, uridine), while others are novel (D-gluconate, N-acetyl-beta-alanine, N-acetylglutamine, orotate, succinate/methylmalonate). The upregulation of the de novo pyrimidine pathway, gluconate shunt and aerobic glycolysis may reflect a wake-dependent need to promote the synthesis of many essential components, from nucleic acids to synaptic membranes.

Metabolomics Approach in the Study of the Well-Defined Polyherbal Preparation Zyflamend.

Zyflamend is a highly controlled blend of 10 herbal extracts that synergistically impact multiple cell signaling pathways with anticancer and anti-inflammatory properties. More recently, its effects were shown to also modify cellular energetics, for example, activation of fatty acid oxidation and inhibition of lipogenesis. However, its general metabolic effects in vivo have yet to be explored. The objective of this study was to characterize the tissue specific metabolomes in response to supplementation of Zyflamend in mice, with a comparison of equivalent metabolomics data generated in plasma from humans supplemented with Zyflamend. Because Zyflamend has been shown to activate AMPK, the "energy sensor" of the cell, in vitro, the effects of Zyflamend on adiposity were also tested in the murine model. C57BL/6 mice were fed diets that mimicked the macro- and micronutrient composition of the U.S. diet with and without Zyflamend supplementation at human equivalent doses. Untargeted metabolomics was performed in liver, skeletal muscle, adipose, and plasma from mice consuming Zyflamend and in plasma from humans supplemented with Zyflamend at an equivalent dose. Adiposity in mice was significantly reduced in the Zyflamend-treated animals (compared with controls) without affecting body weight or weight gain. Based on KEGG pathway enrichment, purine and pyrimidine metabolism (potential regulators of AMPK) were particularly responsive to Zyflamend across all tissues, but only in mice. Consistent with the metabolomics data, Zyflamend activated AMPK and inhibited acetyl CoA-carboxylase in adipose tissue, key regulators of lipogenesis. Zyflamend reduces adipose tissue in mice through a mechanism that likely involves the activation of AMPK.

Maternal consumption of fish oil programs reduced adiposity in broiler chicks.

Maternal intake of eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (22:6 n-3) has been associated with reduced adiposity in children, suggesting the possibility to program adipose development through dietary fatty acids before birth. This study determined if enriching the maternal diet in fish oil, the primary source of EPA and DHA, affected adipose development in offspring. Broiler chickens were used because they are obesity-prone, and because fatty acids provided to the embryo can be manipulated through the hen diet. Hens were fed diets supplemented (2.8% wt:wt) with corn oil (CO; n-6) or fish oil (FO; n-3) for 28 d. Chicks from both maternal diet groups were fed the same diet after hatch. Maternal FO consumption enriched chick adipose tissue in EPA and DHA and reduced adiposity by promoting more, but smaller, adipocytes. This adipocyte profile was paralleled by upregulated expression of the adipogenic regulator PPARG and its co-activator PPARGC1B, and reduced expression of LPL. Proteomics identified 95 differentially abundant proteins between FO and CO adipose tissue, including components of glucose metabolism, lipid droplet trafficking, and cytoskeletal organization. These results demonstrate that the maternal dietary fatty acid profile programs offspring adipose development.

Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in Microcystis.

The cyanobacterium Microcystis aeruginosa is a globally distributed bloom-forming organism that degrades freshwater systems around the world. Factors that drive its dispersion, diversification and success remain, however, poorly understood. To develop insight into cellular-level responses to nutrient drivers of eutrophication, RNA sequencing was coupled to a comprehensive metabolomics survey of M. aeruginosa sp. NIES 843 grown in various nutrient-reduced conditions. Transcriptomes were generated for cultures grown in nutrient-replete (with nitrate as the nitrogen (N) source), nitrogen-reduced (with nitrate, urea or ammonium acting as the N sources) and phosphate-reduced conditions. Extensive expression differences (up to 696 genes for urea-grown cells) relative to the control treatment were observed, demonstrating that the chemical variant of nitrogen available to cells affected transcriptional activity. Of particular note, a high number of transposase genes (up to 81) were significantly and reproducibly up-regulated relative to the control when grown on urea. Conversely, phosphorus (P) reduction resulted in a significant cessation in transcription of transposase genes, indicating that variation in nutrient chemistry may influence transcription of transposases and may impact the highly mosaic genomic architecture of M. aeruginosa. Corresponding metabolomes showed comparably few differences between treatments, suggesting broad changes to gene transcription are required to maintain metabolic homeostasis under nutrient reduction. The combined observations provide novel and extensive insight into the complex cellular interactions that take place in this important bloom-forming organism during variable nutrient conditions and highlight a potential unknown molecular mechanism that may drive Microcystis blooms and evolution.

Quorum sensing control of phosphorus acquisition in Trichodesmium consortia.

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO(2) and N(2), have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)-the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules-led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.