A subpopulation of lipogenic brown adipocytes drives thermogenic memory.

Sustained responses to transient environmental stimuli are important for survival. The mechanisms underlying long-term adaptations to temporary shifts in abiotic factors remain incompletely understood. Here, we find that transient cold exposure leads to sustained transcriptional and metabolic adaptations in brown adipose tissue, which improve thermogenic responses to secondary cold encounter. Primary thermogenic challenge triggers the delayed induction of a lipid biosynthesis programme even after cessation of the original stimulus, which protects from subsequent exposures. Single-nucleus RNA sequencing and spatial transcriptomics reveal that this response is driven by a lipogenic subpopulation of brown adipocytes localized along the perimeter of Ucp1hi adipocytes. This lipogenic programme is associated with the production of acylcarnitines, and supplementation of acylcarnitines is sufficient to recapitulate improved secondary cold responses. Overall, our data highlight the importance of heterogenous brown adipocyte populations for 'thermogenic memory', which may have therapeutic implications for leveraging short-term thermogenesis to counteract obesity.

Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

Myocardial Metabolomics of Human Heart Failure With Preserved Ejection Fraction.

BACKGROUND: The human heart primarily metabolizes fatty acids, and this decreases as alternative fuel use rises in heart failure with reduced ejection fraction (HFrEF). Patients with severe obesity and diabetes are thought to have increased myocardial fatty acid metabolism, but whether this is found in those who also have heart failure with preserved ejection fraction (HFpEF) is unknown. METHODS: Plasma and endomyocardial biopsies were obtained from HFpEF (n=38), HFrEF (n=30), and nonfailing donor controls (n=20). Quantitative targeted metabolomics measured organic acids, amino acids, and acylcarnitines in myocardium (72 metabolites) and plasma (69 metabolites). The results were integrated with reported RNA sequencing data. Metabolomics were analyzed using agnostic clustering tools, Kruskal-Wallis test with Dunn test, and machine learning. RESULTS: Agnostic clustering of myocardial but not plasma metabolites separated disease groups. Despite more obesity and diabetes in HFpEF versus HFrEF (body mass index, 39.8 kg/m2 versus 26.1 kg/m2; diabetes, 70% versus 30%; both P<0.0001), medium- and long-chain acylcarnitines (mostly metabolites of fatty acid oxidation) were markedly lower in myocardium from both heart failure groups versus control. In contrast, plasma levels were no different or higher than control. Gene expression linked to fatty acid metabolism was generally lower in HFpEF versus control. Myocardial pyruvate was higher in HFpEF whereas the tricarboxylic acid cycle intermediates succinate and fumarate were lower, as were several genes controlling glucose metabolism. Non-branched-chain and branched-chain amino acids (BCAA) were highest in HFpEF myocardium, yet downstream BCAA metabolites and genes controlling BCAA metabolism were lower. Ketone levels were higher in myocardium and plasma of patients with HFrEF but not HFpEF. HFpEF metabolomic-derived subgroups were differentiated by only a few differences in BCAA metabolites. CONCLUSIONS: Despite marked obesity and diabetes, HFpEF myocardium exhibited lower fatty acid metabolites compared with HFrEF. Ketones and metabolites of the tricarboxylic acid cycle and BCAA were also lower in HFpEF, suggesting insufficient use of alternative fuels. These differences were not detectable in plasma and challenge conventional views of myocardial fuel use in HFpEF with marked diabetes and obesity and suggest substantial fuel inflexibility in this syndrome.

Stable isotope tracing in vivo reveals a metabolic bridge linking the microbiota to host histone acetylation.

The gut microbiota influences acetylation on host histones by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-coenzyme A (CoA), a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.

Defects in the Proteome and Metabolome in Human Hypertrophic Cardiomyopathy.

BACKGROUND: Defects in energetics are thought to be central to the pathophysiology of hypertrophic cardiomyopathy (HCM); yet, the determinants of ATP availability are not known. The purpose of this study is to ascertain the nature and extent of metabolic reprogramming in human HCM, and its potential impact on contractile function. METHODS: We conducted proteomic and targeted, quantitative metabolomic analyses on heart tissue from patients with HCM and from nonfailing control human hearts. RESULTS: In the proteomic analysis, the greatest differences observed in HCM samples compared with controls were increased abundances of extracellular matrix and intermediate filament proteins and decreased abundances of muscle creatine kinase and mitochondrial proteins involved in fatty acid oxidation. These differences in protein abundance were coupled with marked reductions in acyl carnitines, byproducts of fatty acid oxidation, in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, branched chain amino acids, and their breakdown products, were all significantly increased in HCM hearts. ATP content, phosphocreatine, nicotinamide adenine dinucleotide and its phosphate derivatives, NADP and NADPH, and acetyl CoA were also severely reduced in HCM compared with control hearts. Functional assays performed on human skinned myocardial fibers demonstrated that the magnitude of observed reduction in ATP content in the HCM samples would be expected to decrease the rate of cross-bridge detachment. Moreover, left atrial size, an indicator of diastolic compliance, was inversely correlated with ATP content in hearts from patients with HCM. CONCLUSIONS: HCM hearts display profound deficits in nucleotide availability with markedly reduced capacity for fatty acid oxidation and increases in ketone bodies and branched chain amino acids. These results have important therapeutic implications for the future design of metabolic modulators to treat HCM.

Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes2. Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.

Metabolic adaptation characterizes short-term resistance to weight loss induced by a low-calorie diet in overweight/obese individuals.

BACKGROUND: Low-calorie diet (LCD)-induced weight loss demonstrates response heterogeneity. Physiologically, a decrease in energy expenditure lower than what is predicted based on body composition (metabolic adaptation) and/or an impaired capacity to increase fat oxidation may hinder weight loss. Understanding the metabolic components that characterize weight loss success is important for optimizing weight loss strategies. OBJECTIVES: We tested the hypothesis that overweight/obese individuals who had lower than expected weight loss in response to a 28-d LCD would be characterized by 1) impaired fat oxidation and 2) whole-body metabolic adaptation. We also characterized the molecular mechanisms associated with weight loss success/failure. METHODS: This was a retrospective comparison of participants who met their predicted weight loss targets [overweight/obese diet sensitive (ODS), n = 23, females = 21, males = 2] and those that did not [overweight/obese diet resistant (ODR), n = 14, females = 12, males = 2] after a 28-d LCD (900-1000 kcal/d). We used whole-body (energy expenditure and fat oxidation) and tissue-specific measurements (metabolic proteins in skeletal muscle, gene expression in adipose tissue, and metabolites in serum) to detect metabolic properties and biomarkers associated with weight loss success. RESULTS: The ODR group had greater mean ± SD metabolic adaptation (-175 ± 149 kcal/d; +119%) than the ODS group (-80 ± 108 kcal/d) after the LCD (P = 0.030). Mean ± SD fat oxidation increased similarly for both groups from baseline (0.0701 ± 0.0206 g/min) to day 28 (0.0869 ± 0.0269 g/min; P < 0.001). A principal component analysis factor comprised of serum 3-hydroxybutyric acid, citrate, leucine/isoleucine, acetyl-carnitine, and 3-hydroxylbutyrlcarnitine was associated with weight loss success at day 28 (std. ß = 0.674, R2 = 0.479, P < 0.001). CONCLUSIONS: Individuals who achieved predicted weight loss targets after a 28-d LCD were characterized by reduced metabolic adaptation. Accumulation of metabolites associated with acetyl-CoA excess and enhanced ketogenesis was identified in the ODS group.This trial was registered at clinicaltrials.gov as NCT01616082.

Multi-omic Analysis of the Interaction between Clostridioides difficile Infection and Pediatric Inflammatory Bowel Disease.

Children with inflammatory bowel diseases (IBD) are particularly vulnerable to infection with Clostridioides difficile (CDI). IBD and IBD + CDI have overlapping symptoms but respond to distinctive treatments, highlighting the need for diagnostic biomarkers. Here, we studied pediatric patients with IBD and IBD + CDI, comparing longitudinal data on the gut microbiome, metabolome, and other measures. The microbiome is dysbiotic and heterogeneous in both disease states, but the metabolome reveals disease-specific patterns. The IBD group shows increased concentrations of markers of inflammation and tissue damage compared with healthy controls, and metabolic changes associate with susceptibility to CDI. In IBD + CDI, we detect both metabolites associated with inflammation/tissue damage and fermentation products produced by C. difficile. The most discriminating metabolite found is isocaproyltaurine, a covalent conjugate of a distinctive C. difficile fermentation product (isocaproate) and an amino acid associated with tissue damage (taurine), which may be useful as a joint marker of the two disease processes.

Correction: Isocitrate dehydrogenase 2 protects mice from high-fat diet-induced metabolic stress by limiting oxidative damage to the mitochondria from brown adipose tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Critical Role for Estrogen-Related Receptor Signaling in Cardiac Maturation.

RATIONALE: The heart undergoes dramatic developmental changes during the prenatal to postnatal transition, including maturation of cardiac myocyte energy metabolic and contractile machinery. Delineation of the mechanisms involved in cardiac postnatal development could provide new insight into the fetal shifts that occur in the diseased heart and unveil strategies for driving maturation of stem cell-derived cardiac myocytes. OBJECTIVE: To delineate transcriptional drivers of cardiac maturation. METHODS AND RESULTS: We hypothesized that ERR (estrogen-related receptor) α and γ, known transcriptional regulators of postnatal mitochondrial biogenesis and function, serve a role in the broader cardiac maturation program. We devised a strategy to knockdown the expression of ERRα and γ in heart after birth (pn-csERRα/γ [postnatal cardiac-specific ERRα/γ]) in mice. With high levels of knockdown, pn-csERRα/γ knockdown mice exhibited cardiomyopathy with an arrest in mitochondrial maturation. RNA sequence analysis of pn-csERRα/γ knockdown hearts at 5 weeks of age combined with chromatin immunoprecipitation with deep sequencing and functional characterization conducted in human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) demonstrated that ERRγ activates transcription of genes involved in virtually all aspects of postnatal developmental maturation, including mitochondrial energy transduction, contractile function, and ion transport. In addition, ERRγ was found to suppress genes involved in fibroblast activation in hearts of pn-csERRα/γ knockdown mice. Disruption of Esrra and Esrrg in mice during fetal development resulted in perinatal lethality associated with structural and genomic evidence of an arrest in cardiac maturation, including persistent expression of early developmental and noncardiac lineage gene markers including cardiac fibroblast signatures. Lastly, targeted deletion of ESRRA and ESRRG in hiPSC-CM derepressed expression of early (transcription factor 21 or TCF21) and mature (periostin, collagen type III) fibroblast gene signatures. CONCLUSIONS: ERRα and γ are critical regulators of cardiac myocyte maturation, serving as transcriptional activators of adult cardiac metabolic and structural genes, an.d suppressors of noncardiac lineages including fibroblast determination.

Isocitrate dehydrogenase 2 protects mice from high-fat diet-induced metabolic stress by limiting oxidative damage to the mitochondria from brown adipose tissue.

Isocitrate dehydrogenase 2 (IDH2) is an NADP+-dependent enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate in the mitochondrial matrix, and is critical for the production of NADPH to limit the accumulation of mitochondrial reactive oxygen species (ROS). Here, we showed that high-fat diet (HFD) feeding resulted in accelerated weight gain in the IDH2KO mice due to a reduction in whole-body energy expenditure. Moreover, the levels of NADP+, NADPH, NAD+, and NADH were significantly decreased in the brown adipose tissue (BAT) of the HFD-fed IDH2KO animals, accompanied by decreased mitochondrial function and reduced expression of key genes involved in mitochondrial biogenesis, energy expenditure, and ROS resolution. Interestingly, these changes were partially reversed when the antioxidant butylated hydroxyanisole was added to the HFD. These observations reveal a crucial role for IDH2 in limiting ROS-dependent mitochondrial damage when BAT metabolism is normally enhanced to limit weight gain in response to dietary caloric overload.

Metabolomics Analyses of Muscle Atrophy Induced by Hind Limb Unloading.

Acute periods of contractile inactivity cause skeletal muscle atrophy along with profound alterations in tissue metabolism. Hind limb unloading via tail suspension is a commonly used rodent model of muscle atrophy. Here, we describe a sample preparation and LC-MS/MS approach for quantifying specific panels of acylcarnitines, amino acids, and organic acids in small (~8 mg) samples of atrophied mouse soleus following a period of hind limb unloading.

The failing heart utilizes 3-hydroxybutyrate as a metabolic stress defense.

Evidence has emerged that the failing heart increases utilization of ketone bodies. We sought to determine whether this fuel shift is adaptive. Mice rendered incapable of oxidizing the ketone body 3-hydroxybutyrate (3OHB) in the heart exhibited worsened heart failure in response to fasting or a pressure overload/ischemic insult compared with WT controls. Increased delivery of 3OHB ameliorated pathologic cardiac remodeling and dysfunction in mice and in a canine pacing model of progressive heart failure. 3OHB was shown to enhance bioenergetic thermodynamics of isolated mitochondria in the context of limiting levels of fatty acids. These results indicate that the heart utilizes 3OHB as a metabolic stress defense and suggest that strategies aimed at increasing ketone delivery to the heart could prove useful in the treatment of heart failure.

Lower Concentrations of Circulating Medium and Long-Chain Acylcarnitines Characterize Insulin Resistance in Persons with HIV.

In human immunodeficiency virus (HIV)-negative individuals, a plasma metabolite profile, characterized by higher levels of branched-chain amino acids (BCAA), aromatic amino acids, and C3/C5 acylcarnitines, is associated with insulin resistance and increased risk of diabetes. We sought to characterize the metabolite profile accompanying insulin resistance in HIV-positive persons to assess whether the same or different bioenergetics pathways might be implicated. We performed an observational cohort study of 70 nondiabetic, HIV-positive individuals (50% with body mass index ≥30 kg/m2) on efavirenz, tenofovir, and emtricitabine with suppressed HIV-1 RNA levels (<50 copies/mL) for at least 2 years and a CD4+ count over 350 cells/µL. We measured fasting insulin resistance using the homeostatic model assessment 2, plasma free fatty acids (FFA) using gas chromatography, and amino acids, acylcarnitines, and organic acids using liquid chromatography/mass spectrometry. We assessed the relationship of plasma metabolites with insulin resistance using multivariable linear regression. The median age was 45 years, median CD4+ count was 701 cells/µL, and median hemoglobin A1c was 5.2%. Insulin resistance was associated with higher plasma C3 acylcarnitines (p = .01), but not BCAA or C5 acylcarnitines. However, insulin resistance was associated with lower plasma levels of C18, C16, C12, and C2 acylcarnitines (p ≤ .03 for all), and lower C18 and C16 acylcarnitine:FFA ratios (p = .002, and p = .03, respectively). In HIV-positive persons, lower levels of plasma acylcarnitines, including the C2 product of complete fatty acid oxidation, are a more prominent feature of insulin resistance than changes in BCAA, suggesting impaired fatty acid uptake and/or mitochondrial oxidation is a central aspect of glucose intolerance in this population.

Targeted Metabolomic Profiling of Plasma and Survival in Heart Failure Patients.

OBJECTIVES: This study sought to derive and validate plasma metabolite associations with survival in heart failure (HF) patients. BACKGROUND: Profiling of plasma metabolites to predict the course of HF appears promising, but validation and incremental value of these profiles are less established. METHODS: Patients (n = 1,032) who met Framingham HF criteria with a history of reduced ejection fraction were randomly divided into derivation and validation cohorts (n = 516 each). Amino acids, organic acids, and acylcarnitines were quantified using mass spectrometry in fasting plasma samples. We derived a prognostic metabolite profile (PMP) in the derivation cohort using Lasso-penalized Cox regression. Validity was assessed by 10-fold cross validation in the derivation cohort and by standard testing in the validation cohort. The PMP was analyzed as both a continuous variable (PMPscore) and dichotomized at the median (PMPcat), in univariate and multivariate models adjusted for clinical risk score and N-terminal pro-B-type natriuretic peptide. RESULTS: Overall, 48% of patients were African American, 35% were women, and the average age was 69 years. After a median follow-up of 34 months, there were 256 deaths (127 and 129 in derivation and validation cohorts, respectively). Optimized modeling defined the 13 metabolite PMPs, which was cross validated as both the PMPscore (hazard ratio [HR]: 3.27; p < 2 × 10-16) and PMPcat (HR: 3.04; p = 2.93 × 10-8). The validation cohort showed similar results (PMPscore HR: 3.9; p < 2 × 10-16 and PMPcat HR: 3.99; p = 3.47 × 10-9). In adjusted models, PMP remained associated with mortality in the cross-validated derivation cohort (PMPscore HR: 1.63; p = 0.0029; PMPcat HR: 1.47; p = 0.081) and the validation cohort (PMPscore HR: 1.54; p = 0.037; PMPcat HR: 1.69; p = 0.043). CONCLUSIONS: Plasma metabolite profiles varied across HF subgroups and were associated with survival incremental to conventional predictors. Additional investigation is warranted to define mechanisms and clinical applications.

Staphylococcus aureus nitric oxide synthase (saNOS) modulates aerobic respiratory metabolism and cell physiology.

Nitric oxide (NO) is generated from arginine and oxygen via NO synthase (NOS). Staphylococcus aureus NOS (saNOS) has previously been shown to affect virulence and resistance to exogenous oxidative stress, yet the exact mechanism is unknown. Herein, a previously undescribed role of saNOS in S. aureus aerobic physiology was reported. Specifically, aerobic S. aureus nos mutant cultures presented with elevated endogenous reactive oxygen species (ROS) and superoxide levels, as well as increased membrane potential, increased respiratory dehydrogenase activity and slightly elevated oxygen consumption. Elevated ROS levels in the nos mutant likely resulted from altered respiratory function, as inhibition of NADH dehydrogenase brought ROS levels back to wild-type levels. These results indicate that, in addition to its recently reported role in regulating the switch to nitrate-based respiration during low-oxygen growth, saNOS also plays a modulatory role during aerobic respiration. Multiple transcriptional changes were also observed in the nos mutant, including elevated expression of genes associated with oxidative/nitrosative stress, anaerobic respiration and lactate metabolism. Targeted metabolomics revealed decreased cellular lactate levels, and altered levels of TCA cycle intermediates, the latter of which may be related to decreased aconitase activity. Collectively, these findings demonstrate a key contribution of saNOS to S. aureus aerobic respiratory metabolism.

Superior Glucose Tolerance and Metabolomic Profiles, Independent of Adiposity, in HIV-Infected Women Compared With Men on Antiretroviral Therapy.

In epidemiologic studies, human immunodeficiency virus (HIV)-infected men on antiretroviral therapy (ART) are at higher risk of incident diabetes mellitus compared with women with similar treatment histories. We used metabolomics to determine whether a sex difference in plasma amino acids, acylcarnitines, and organic acids predictive of diabetes and impaired energy metabolism is present in HIV-infected persons on long-term ART.We enrolled 70 HIV-infected adults (43% women) on efavirenz, tenofovir, and emtricitabine (Atripla) with HIV-1 RNA <50 copies/mL for over 2 years. Half of the HIV-infected subjects were obese, and these were matched with 30 obese HIV-negative controls. All subjects had no history of diabetes, statin use, or heavy alcohol use. Fasting insulin sensitivity was measured using homeostatic model assessment 2 (HOMA2), and adipose tissue was measured using dual-energy x-ray absorptiometry (DEXA). Liquid chromatography/mass spectrometry was used to quantitate fasting plasma branched chain and aromatic amino acids predictive of incident diabetes, and C3 and C5 acylcarnitinines and organic acids indicative of impaired energy metabolism.HIV-infected women had more baseline risk factors for insulin resistance: women were older (46 vs 44 years) and had a longer ART duration (8.4 vs 5.1 years, P < 0.05 for both) compared with men but had similar CD4+ count (median 701 cells/µL), smoking and hepatic C prevalence, and body mass index (BMI) (median 30.3 kg/m). However, women had higher insulin sensitivity compared with men (P < 0.01), and lower plasma levels of isoleucine, leucine, valine, phenylalanine, and tyrosine (P < 0.01 for all), and lower C3 and C5 acylcarnitines (P < 0.01 for all), in multivariable regression models after adjusting for DEXA fat mass index, age, race, CD4+ count, smoking, and ART duration. In the obese HIV-infected subjects and HIV-negative controls, the relationship of sex and plasma metabolite levels did not significantly differ according to HIV-status.HIV-infected women on non-nucleoside reverse transcriptase inhibitor-based ART had superior glucose tolerance and lower plasma metabolites associated with the development of diabetes compared with men with similar metabolic disease risk profiles. The relationship between sex and plasma metabolite levels did not significantly differ according to HIV-status among obese subjects, suggesting the observed sex-differences may not be specific to HIV infection.

Presence of arachidonoyl-carnitine is associated with adverse cardiometabolic responses in hypertensive patients treated with atenolol.

INTRODUCTION: Atenolol, a commonly prescribed ß blocker for hypertension, is also associated with adverse cardiometabolic effects such as hyperglycemia and dyslipidemia. Knowledge of the mechanistic underpinnings of these adverse effects of atenolol is incomplete. OBJECTIVE: We sought to identify biomarkers associated with risk for these untoward effects of atenolol. We measured baseline blood serum levels of acylcarnitines (ACs) that are involved in a host of different metabolic pathways, to establish associations with adverse cardiometabolic responses after atenolol treatment. METHODS: Serum samples from Caucasian hypertensive patients (n = 224) who were treated with atenolol in the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study were interrogated using a quantitative LC/MS assay for a large number of unique ACs in serum. For the 23 ACs that were detected in serum from ≥80 % of all patients, we conducted linear regression for changes in cardiometabolic factors with baseline AC levels, baseline cardiometabolic factors, age, sex, and BMI as covariates. For the 5 ACs that were detected in serum from 20 to 79 % of the patients, we similarly modeled changes in cardiometabolic factors, but with specifying the AC as present/absent in the regression. RESULTS: Among the 28 ACs, the presence (vs. absence) of arachidonoyl-carnitine (C20:4) was significantly associated with increased glucose (p = 0.0002), and was nominally associated with decreased plasma HDL-C (p = 0.017) and with less blood pressure (BP) lowering (p = 0.006 for systolic BP, p = 0.002 for diastolic BP), after adjustment. CONCLUSION: Serum level of C20:4 is a promising biomarker to predict adverse cardiometabolic responses including glucose and poor antihypertensive response to atenolol.

Rotenone induces reductive stress and triacylglycerol deposition in C2C12 cells.

Environmental rotenone is associated with Parkinson's disease due to its inhibitory property to the complex I of mitochondrial respiration chain. Although environmental pollution has been postulated as a causal factor for the increasing prevalence of obesity, the role of rotenone in the pathogenesis of obesity has not been studied. We employed muscle-derived cell C2C12 as a model and shotgun lipidomics as a tool for lipid analysis and found that treatment with rotenone led to the profound deposition of intracellular triacylglycerol (TAG) in a time- and dose-dependent fashion. The TAG deposition resulted from complex I inhibition. Further studies revealed that rotenone induced mitochondrial stress as shown by decreased mitochondrial oxygen consumption rate, increased NADH/NAD+ ratio (i.e., reductive stress) and mitochondrial metabolites. We demonstrated that rotenone activated fatty acid de novo synthesis and TAG synthesis and ultimately resulted in intracellular TAG deposition. These studies suggested that increased mitochondrial stresses might be an underlying mechanism responsible for TAG accumulation manifest in obesity.

Direct and quantitative analysis of underivatized acylcarnitines in serum and whole blood using paper spray mass spectrometry.

A simple protocol for rapid quantitation of acylcarnitines in serum and whole blood has been developed using paper spray mass spectrometry. Dried serum and whole blood containing a mixture of ten acylcarnitines at various concentrations were analyzed as spots from paper directly without any sample pretreatment, separation, or derivatization. The composition of the spray solvent was found to be a critical factor: for serum samples, spray solvent of methanol/water/formic acid (80:20:0.1) gave the best signal intensity while for blood samples which contain more matrix components, acetonitrile/water (90:10) was a much more suitable spray solvent. For the paper type and size used, 0.5 µL of sample provided an optimal signal for both serum and whole blood samples. For quantitative profiling, the limits of quantitation obtained from both serum and blood were much lower than the clinically validated cutoff values for diagnosis of fatty acid oxidation disorders in newborn screening. Linearity (R(2) > 0.95) and reproducibility (RSD ~10 %) were achieved in the concentration ranges from 100 nM to 5 µM for the C2 acylcarnitine, and for other acylcarnitines, these values were from 10 to 500 nM. Acylcarnitine profiles offer an effective demonstration of the fact that paper spray mass spectrometry is an appropriate, simple, rapid method with high sensitivity and high reproducibility applicable to newborn screening tests.