Demographic, Health and Lifestyle Factors Associated with the Metabolome in Older Women.

Demographic and clinical factors influence the metabolome. The discovery and validation of disease biomarkers are often challenged by potential confounding effects from such factors. To address this challenge, we investigated the magnitude of the correlation between serum and urine metabolites and demographic and clinical parameters in a well-characterized observational cohort of 444 post-menopausal women participating in the Women's Health Initiative (WHI). Using LC-MS and lipidomics, we measured 157 aqueous metabolites and 756 lipid species across 13 lipid classes in serum, along with 195 metabolites detected by GC-MS and NMR in urine and evaluated their correlations with 29 potential disease risk factors, including demographic, dietary and lifestyle factors, and medication use. After controlling for multiple testing (FDR < 0.01), we found that log-transformed metabolites were mainly associated with age, BMI, alcohol intake, race, sample storage time (urine only), and dietary supplement use. Statistically significant correlations were in the absolute range of 0.2-0.6, with the majority falling below 0.4. Incorporation of important potential confounding factors in metabolite and disease association analyses may lead to improved statistical power as well as reduced false discovery rates in a variety of data analysis settings.

Cerebrospinal Fluid Metabolomics: Pilot Study of Using Metabolomics to Assess Diet and Metabolic Interventions in Alzheimer's Disease and Mild Cognitive Impairment.

Brain glucose hypometabolism is an early sign of Alzheimer's disease (AD), and interventions which offset this deficit, such as ketogenic diets, show promise as AD therapeutics. Conversely, high-fat feeding may exacerbate AD risk. We analyzed the metabolomic profile of cerebrospinal fluid (CSF) in a pilot study of older adults who underwent saline and triglyceride (TG) infusions. Older adults (12 cognitively normal (CN), age 65.3 ± 8.1, and 9 with cognitive impairment (CI), age 70.9 ± 8.6) underwent a 5 h TG or saline infusion on different days using a random crossover design; CSF was collected at the end of infusion. Aqueous metabolites were measured using a targeted mass spectroscopy (MS) platform focusing on 215 metabolites from over 35 different metabolic pathways. Data were analyzed using MetaboAnalyst 4.0 and SAS. Of the 215 targeted metabolites, 99 were detectable in CSF. Only one metabolite significantly differed by treatment: the ketone body 3-hydroxybutyrate (HBA). Post hoc analyses showed that HBA levels were associated with age and markers of metabolic syndrome and demonstrated different correlation patterns for the two treatments. When analyzed by cognitive diagnosis group, TG-induced increases in HBA were over 3 times higher for those with cognitive impairment (change score CN +9.8 uM ± 8.3, CI +32.4 ± 7.4, p = 0.0191). Interestingly, individuals with cognitive impairment had higher HBA levels after TG infusion than those with normal cognition. These results suggest that interventions that increase plasma ketones may lead to higher brain ketones in groups at risk for AD and should be confirmed in larger intervention studies.

A DMS Shotgun Lipidomics Workflow Application to Facilitate High-Throughput, Comprehensive Lipidomics.

Differential mobility spectrometry (DMS) is highly useful for shotgun lipidomic analysis because it overcomes difficulties in measuring isobaric species within a complex lipid sample and allows for acyl tail characterization of phospholipid species. Despite these advantages, the resulting workflow presents technical challenges, including the need to tune the DMS before every batch to update compensative voltages settings within the method. The Sciex Lipidyzer platform uses a Sciex 5500 QTRAP with a DMS (SelexION), an LC system configured for direction infusion experiments, an extensive set of standards designed for quantitative lipidomics, and a software package (Lipidyzer Workflow Manager) that facilitates the workflow and rapidly analyzes the data. Although the Lipidyzer platform remains very useful for DMS-based shotgun lipidomics, the software is no longer updated for current versions of Analyst and Windows. Furthermore, the software is fixed to a single workflow and cannot take advantage of new lipidomics standards or analyze additional lipid species. To address this multitude of issues, we developed Shotgun Lipidomics Assistant (SLA), a Python-based application that facilitates DMS-based lipidomics workflows. SLA provides the user with flexibility in adding and subtracting lipid and standard MRMs. It can report quantitative lipidomics results from raw data in minutes, comparable to the Lipidyzer software. We show that SLA facilitates an expanded lipidomics analysis that measures over 1450 lipid species across 17 (sub)classes. Lastly, we demonstrate that the SLA performs isotope correction, a feature that was absent from the original software.

Depletion of mitochondrial inorganic polyphosphate (polyP) in mammalian cells causes metabolic shift from oxidative phosphorylation to glycolysis.

Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical with those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.

Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles.

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson's disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. Accordingly, we hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. We found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread.

Persistent metabolomic alterations characterize chronic critical illness after severe trauma.

BACKGROUND: Following trauma, persistent inflammation, immunosuppression, and catabolism may characterize delayed recovery or failure to recover. Understanding the metabolic response associated with these adverse outcomes may facilitate earlier identification and intervention. We characterized the metabolic profiles of trauma victims who died or developed chronic critical illness (CCI) and hypothesized that differences would be evident within 1-week postinjury. METHODS: Venous blood samples from trauma victims with shock who survived at least 7 days were analyzed using mass spectrometry. Subjects who died or developed CCI (intensive care unit length of stay of ≥14 days with persistent organ dysfunction) were compared with subjects who recovered rapidly (intensive care unit length of stay, ≤7 days) and uninjured controls. We used partial least squares discriminant analysis, t tests, linear mixed effects regression, and pathway enrichment analyses to make broad comparisons and identify differences in metabolite concentrations and pathways. RESULTS: We identified 27 patients who died or developed CCI and 33 who recovered rapidly. Subjects were predominantly male (65%) with a median age of 53 years and Injury Severity Score of 36. Healthy controls (n = 48) had similar age and sex distributions. Overall, from the 163 metabolites detected in the samples, 56 metabolites and 21 pathways differed between injury outcome groups, and partial least squares discriminant analysis models distinguished injury outcome groups as early as 1-day postinjury. Differences were observed in tryptophan, phenylalanine, and tyrosine metabolism; metabolites associated with oxidative stress via methionine metabolism; inflammatory mediators including kynurenine, arachidonate, and glucuronic acid; and products of the gut microbiome including indole-3-propionate. CONCLUSIONS: The metabolic profiles in subjects who ultimately die or develop CCI differ from those who have recovered. In particular, we have identified differences in markers of inflammation, oxidative stress, amino acid metabolism, and alterations in the gut microbiome. Targeted metabolomics has the potential to identify important metabolic changes postinjury to improve early diagnosis and targeted intervention. LEVEL OF EVIDENCE: Prognostic/epidemiologic, level III.

Plasma lipidomic profiles after a low and high glycemic load dietary pattern in a randomized controlled crossover feeding study.

BACKGROUND: Dietary patterns low in glycemic load are associated with reduced risk of cardiometabolic diseases. Improvements in serum lipid concentrations may play a role in these observed associations. OBJECTIVE: We investigated how dietary patterns differing in glycemic load affect clinical lipid panel measures and plasma lipidomics profiles. METHODS: In a crossover, controlled feeding study, 80 healthy participants (n = 40 men, n = 40 women), 18-45 y were randomized to receive low-glycemic load (LGL) or high glycemic load (HGL) diets for 28 days each with at least a 28-day washout period between controlled diets. Fasting plasma samples were collected at baseline and end of each diet period. Lipids on a clinical panel including total-, VLDL-, LDL-, and HDL-cholesterol and triglycerides were measured using an auto-analyzer. Lipidomics analysis using mass-spectrometry provided the concentrations of 863 species. Linear mixed models and lipid ontology enrichment analysis were implemented. RESULTS: Lipids from the clinical panel were not significantly different between diets. Univariate analysis showed that 67 species on the lipidomics panel, predominantly in the triacylglycerol class, were higher after the LGL diet compared to the HGL (FDR < 0.05). Three species with FA 17:0 were lower after LGL diet with enrichment analysis (FDR < 0.05). CONCLUSION: In the context of controlled eucaloric diets with similar macronutrient distribution, these results suggest that there are relative shifts in lipid species, but the overall pool does not change. Further studies are needed to better understand in which compartment the different lipid species are transported in blood, and how these shifts are related to health outcomes. This trial was registered at clinicaltrials.gov as NCT00622661.

Metabolomic profiling for juvenile Chinook salmon exposed to contaminants of emerging concern.

Both targeted and non-targeted metabolomic analyses were conducted on juvenile ocean-type fall Chinook salmon (Oncorhynchus tshawytscha) residing in two estuaries receiving wastewater treatment plant (WWTP) effluent and one reference estuary. The data show that the metabolome patterns for fish from the two WWTP-receiving estuaries were more similar to each other compared to that for the reference site fish. Also, a comparison of the metabolome for fish from the reference site and fish from a hatchery upstream of one of the effluent-receiving estuaries indicated no differences, implying that residency for fish in the contaminated estuary resulted in major changes to the metabolome. Based on general health parameters including whole-body lipid content and condition factor, plus the availability of prey for these fish, we conclude that juvenile Chinook salmon in these contaminated estuaries may have been experiencing metabolic disruption without any overt signs of impairment. Additionally, a non-targeted analysis was performed on hatchery summer Chinook salmon from a laboratory study where fish were dosed for 32 days with feed containing 16 of the most common contaminants of emerging concern (CECs) detected in wild fish. In the laboratory experiment a relationship was observed between dose and the number of liver metabolites that were different between control and treatment fish. Laboratory fish were exposed to only 16 CECs, but are generally exposed to hundreds of these compounds in contaminated aquatic environments. These results have implications for the health of juvenile Chinook salmon and the likelihood of a successful life cycle when exposed to effluent-related chemicals.

Increased Drp1 Acetylation by Lipid Overload Induces Cardiomyocyte Death and Heart Dysfunction.

RATIONALE: Lipid overload-induced heart dysfunction is characterized by cardiomyocyte death, myocardial remodeling, and compromised contractility, but the impact of excessive lipid supply on cardiac function remains poorly understood. OBJECTIVE: To investigate the regulation and function of the mitochondrial fission protein Drp1 (dynamin-related protein 1) in lipid overload-induced cardiomyocyte death and heart dysfunction. METHODS AND RESULTS: Mice fed a high-fat diet (HFD) developed signs of obesity and type II diabetes mellitus, including hyperlipidemia, hyperglycemia, hyperinsulinemia, and hypertension. HFD for 18 weeks also induced heart hypertrophy, fibrosis, myocardial insulin resistance, and cardiomyocyte death. HFD stimulated mitochondrial fission in mouse hearts. Furthermore, HFD increased the protein level, phosphorylation (at the activating serine 616 sites), oligomerization, mitochondrial translocation, and GTPase activity of Drp1 in mouse hearts, indicating that Drp1 was activated. Monkeys fed a diet high in fat and cholesterol for 2.5 years also exhibited myocardial damage and Drp1 activation in the heart. Interestingly, HFD decreased nicotinamide adenine dinucleotide (oxidized) levels and increased Drp1 acetylation in the heart. In adult cardiomyocytes, palmitate increased Drp1 acetylation, phosphorylation, and protein levels, and these increases were abolished by restoration of the decreased nicotinamide adenine dinucleotide (oxidized) level. Proteomics analysis and in vitro screening revealed that Drp1 acetylation at lysine 642 (K642) was increased by HFD in mouse hearts and by palmitate incubation in cardiomyocytes. The nonacetylated Drp1 mutation (K642R) attenuated palmitate-induced Drp1 activation, its interaction with voltage-dependent anion channel 1, mitochondrial fission, contractile dysfunction, and cardiomyocyte death. CONCLUSIONS: These findings uncover a novel mechanism that contributes to lipid overload-induced heart hypertrophy and dysfunction. Excessive lipid supply created an intracellular environment that facilitated Drp1 acetylation, which, in turn, increased its activity and mitochondrial translocation, resulting in cardiomyocyte dysfunction and death. Thus, Drp1 may be a critical mediator of lipid overload-induced heart dysfunction as well as a potential target for therapy.

Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis.

RATIONALE: 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. OBJECTIVE: The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Cerebrospinal fluid lipidomics: effects of an intravenous triglyceride infusion and apoE status.

INTRODUCTION: High-fat diets increase risk for Alzheimer's disease, but individuals with the risk gene APOE ε4 (E4) paradoxically have improved memory soon after high fat feeding. Little is known about how dietary lipids affect CNS lipids, especially in older adults. OBJECTIVES: We analyzed the lipidomic signature of cerebrospinal fluid (CSF) in older adults who underwent both a saline and TG infusion. We further analyzed these data by E4 carrier status. METHODS: Older adults (n = 21, age 67.7 ± 8.6) underwent a 5-h TG and saline infusion on different days in random crossover design; lumbar CSF was collected at the end of the infusion. Lipids were extracted using dichloromethane/methanol and 13 classes of lipids analyzed using the Lipidyzer platform consisting of an AB Sciex 5500 MS/MS QTraps system equipped with a SelexION for differential mobility spectrometry (DMS). Multiple reaction monitoring was used to target and quantify 1070 lipids in positive and negative ionization modes with and without DMS. RESULTS: The TG infusion increased total lipids in the CSF, including the appearance of more lipids at the detection limit in the TG samples compared to saline (Chi square p < 0.0001). The infusion increased the total level of diacylglycerols and lysophosphatidylcholines and reduced dihydroceramides. Of the possible 1070 lipids detectable, we found 348 after saline and 365 after TG infusion. Analysis using MetaboAnalyst revealed 11 specific lipids that changed; five of these lipids decreased after TG infusion, and four of them differed by E4 status, but none differed by cognitive diagnosis or sex. CONCLUSION: These results in older adults show that blood lipids affect lipid profiles in CSF and such profiles are modified by APOE status. This suggests that how the CNS handles lipids may be important in the AD phenotype.