Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.

Mitochondrial pyruvate carrier inhibitors improve metabolic parameters in diet-induced obese mice.

The mitochondrial pyruvate carrier (MPC) is an inner mitochondrial membrane complex that plays a critical role in intermediary metabolism. Inhibition of the MPC, especially in liver, may have efficacy for treating type 2 diabetes mellitus. Herein, we examined the antidiabetic effects of zaprinast and 7ACC2, small molecules which have been reported to act as MPC inhibitors. Both compounds activated a bioluminescence resonance energy transfer-based MPC reporter assay (reporter sensitive to pyruvate) and potently inhibited pyruvate-mediated respiration in isolated mitochondria. Furthermore, zaprinast and 7ACC2 acutely improved glucose tolerance in diet-induced obese mice in vivo. Although some findings were suggestive of improved insulin sensitivity, hyperinsulinemic-euglycemic clamp studies did not detect enhanced insulin action in response to 7ACC2 treatment. Rather, our data suggest acute glucose-lowering effects of MPC inhibition may be due to suppressed hepatic gluconeogenesis. Finally, we used reporter sensitive to pyruvate to screen a chemical library of drugs and identified 35 potentially novel MPC modulators. Using available evidence, we generated a pharmacophore model to prioritize which hits to pursue. Our analysis revealed carsalam and six quinolone antibiotics, as well as 7ACC1, share a common pharmacophore with 7ACC2. We validated that these compounds are novel inhibitors of the MPC and suppress hepatocyte glucose production and demonstrated that one quinolone (nalidixic acid) improved glucose tolerance in obese mice. In conclusion, these data demonstrate the feasibility of therapeutic targeting of the MPC for treating diabetes and provide scaffolds that can be used to develop potent and novel classes of MPC inhibitors.

Hyperammonaemia disrupts daily rhythms reversibly by elevating glutamate in the central circadian pacemaker.

Patients with cirrhosis exhibit features of circadian disruption. Hyperammonaemia has been suggested to impair both homeostatic and circadian sleep regulation. Here, we tested if hyperammonaemia directly disrupts circadian rhythm generation in the central pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus. Wheel-running activity was recorded from mice fed with a hyperammonaemic or normal diet for ~35 days in a 12:12 light-dark (LD) cycle followed by ~15 days in constant darkness (DD). The expression of the clock protein PERIOD2 (PER2) was recorded from SCN explants before, during and after ammonia exposure, ±glutamate receptor antagonists. In LD, hyperammonaemic mice advanced their daily activity onset time by ~1 h (16.8 ± 0.3 vs. 18.1 ± 0.04 h, p = .009) and decreased their total activity, concentrating it during the first half of the night. In DD, hyperammonaemia reduced the amplitude of daily activity (551.5 ± 27.7 vs. 724.9 ± 59 counts, p = .007), with no changes in circadian period. Ammonia (≥0.01 mM) rapidly and significantly reduced PER2 amplitude, and slightly increased circadian period. The decrease in PER2 amplitude correlated with decreased synchrony among circadian cells in the SCN and increased extracellular glutamate, which was rescued by AMPA glutamate receptor antagonists. These data suggest that hyperammonaemia affects circadian regulation of rest-activity behaviour by increasing extracellular glutamate in the SCN.

Race Is Associated With Risk of Salvage Procedures and Postoperative Complications After Hip Procedures in Children With Cerebral Palsy.

BACKGROUND: Despite the many surgical interventions available for spastic hip dysplasia in children with cerebral palsy, a radical salvage hip procedure may still ultimately be required. The purpose of this study was to assess whether race is an independent risk factor for patients with cerebral palsy to undergo a salvage hip procedure or experience postoperative complications for hip dysplasia treatment. METHODS: This is a retrospective cohort analysis utilizing the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) Pediatric database from 2012 to 2019. International Classification of Diseases, 9th and 10th Revisions, Clinical Modifications (ICD-9-CM, ICD-10-CM), and current procedural terminology (CPT) codes were used to identify patients with cerebral palsy undergoing hip procedures for hip dysplasia and to stratify patients into salvage or reconstructive surgeries. RESULTS: There was a total of 3906 patients with cerebral palsy between the ages of 2 and 18 years undergoing a procedure for hip dysplasia, including 1995 (51.1%) White patients, 768 (19.7%) Black patients, and 1143 (29.3%) patients from other races. Both Black ( P =0.044) and White ( P =0.046) races were significantly associated with undergoing a salvage versus a reconstructive hip procedure, with Black patients having an increased risk compared to White patients [adjusted odds ratio (OR) 1.77, confidence interval (CI) 1.02-3.07]. Only Black patients were found to have an increased risk of any postoperative complication compared to White patients, with an adjusted OR of 1.26 (CI 1.02-1.56; P =0.033). Both White ( P =0.017) and black ( P =0.004) races were found to be significantly associated with medical complications, with Black patients having an increased risk (adjusted OR 1.43, CI 1.12-1.84) compared to White patients. There were no significant findings between the race and risk of surgical site complications, unplanned readmissions, or reoperations. CONCLUSION: This study demonstrates that patient race is an independent association for the risk of pediatric patients with cerebral palsy to both undergo a salvage hip procedure and to experience postoperative medical complications, with Black patients having an increased risk compared to White. LEVEL OF EVIDENCE: Level III Retrospective Cohort Study.

Dose-Response Metabolomics To Understand Biochemical Mechanisms and Off-Target Drug Effects with the TOXcms Software.

Small-molecule drugs and toxicants commonly interact with more than a single protein target, each of which may have unique effects on cellular phenotype. Although untargeted metabolomics is often applied to understand the mode of action of these chemicals, simple pairwise comparisons of treated and untreated samples are insufficient to resolve the effects of disrupting two or more independent protein targets. Here, we introduce a workflow for dose-response metabolomics to evaluate chemicals that potentially affect multiple proteins with different potencies. Our approach relies on treating samples with various concentrations of compound prior to analysis with mass spectrometry-based metabolomics. Data are then processed with software we developed called TOXcms, which statistically evaluates dose-response trends for each metabolomic signal according to user-defined tolerances and subsequently groups those that follow the same pattern. Although TOXcms was built upon the XCMS framework, it is compatible with any metabolomic data-processing software. Additionally, to enable correlation of dose responses beyond those that can be measured by metabolomics, TOXcms also accepts data from respirometry, cell death assays, other omic platforms, etc. In this work, we primarily focus on applying dose-response metabolomics to find off-target effects of drugs. Using metformin and etomoxir as examples, we demonstrate that each group of dose-response patterns identified by TOXcms signifies a metabolic response to a different protein target with a unique drug binding affinity. TOXcms is freely available on our laboratory website at http://pattilab.wustl.edu/software/toxcms .

Bar Coding MS(2) Spectra for Metabolite Identification.

Metabolite identifications are most frequently achieved in untargeted metabolomics by matching precursor mass and full, high-resolution MS(2) spectra to metabolite databases and standards. Here we considered an alternative approach for establishing metabolite identifications that does not rely on full, high-resolution MS(2) spectra. First, we select mass-to-charge regions containing the most informative metabolite fragments and designate them as bins. We then translate each metabolite fragmentation pattern into a binary code by assigning 1's to bins containing fragments and 0's to bins without fragments. With 20 bins, this binary-code system is capable of distinguishing 96% of the compounds in the METLIN MS(2) library. A major advantage of the approach is that it extends untargeted metabolomics to low-resolution triple quadrupole (QqQ) instruments, which are typically less expensive and more robust than other types of mass spectrometers. We demonstrate a method of acquiring MS(2) data in which the third quadrupole of a QqQ instrument cycles over 20 wide isolation windows (coinciding with the location and width of our bins) for each precursor mass selected by the first quadrupole. Operating the QqQ instrument in this mode yields diagnostic bar codes for each precursor mass that can be matched to the bar codes of metabolite standards. Furthermore, our data suggest that using low-resolution bar codes enables QqQ instruments to make MS(2)-based identifications in untargeted metabolomics with a specificity and sensitivity that is competitive to high-resolution time-of-flight technologies.

Differential incorporation of glucose into biomass during Warburg metabolism.

It is well established that most cancer cells take up an increased amount of glucose relative to that taken up by normal differentiated cells. The majority of this glucose carbon is secreted from the cell as lactate. The fate of the remaining glucose carbon, however, has not been well-characterized. Here we apply a novel combination of metabolomic technologies to track uniformly labeled glucose in HeLa cancer cells. We provide a list of specific intracellular metabolites that become enriched after being labeled for 48 h and quantitate the fraction of consumed glucose that ends up in proteins, peptides, sugars/glycerol, and lipids.

isoMETLIN: a database for isotope-based metabolomics.

The METLIN metabolite database has become one of the most widely used resources in metabolomics for making metabolite identifications. However, METLIN is not designed to identify metabolites that have been isotopically labeled. As a result, unbiasedly tracking the transformation of labeled metabolites with isotope-based metabolomics is a challenge. Here, we introduce a new database, called isoMETLIN (http://isometlin.scripps.edu/), that has been developed specifically to identify metabolites incorporating isotopic labels. isoMETLIN enables users to search all computed isotopologues derived from METLIN on the basis of mass-to-charge values and specified isotopes of interest, such as (13)C or (15)N. Additionally, isoMETLIN contains experimental MS/MS data on hundreds of isotopomers. These data assist in localizing the position of isotopic labels within a metabolite. From these experimental MS/MS isotopomer spectra, precursor atoms can be mapped to fragments. The MS/MS spectra of additional isotopomers can then be computationally generated and included within isoMETLIN. Given that isobaric isotopomers cannot be separated chromatographically or by mass but are likely to occur simultaneously in a biological system, we have also implemented a spectral-mixing function in isoMETLIN. This functionality allows users to combine MS/MS spectra from various isotopomers in different ratios to obtain a theoretical MS/MS spectrum that matches the MS/MS spectrum from a biological sample. Thus, by searching MS and MS/MS experimental data, isoMETLIN facilitates the identification of isotopologues as well as isotopomers from biological samples and provides a platform to drive the next generation of isotope-based metabolomic studies.

X13CMS: global tracking of isotopic labels in untargeted metabolomics.

Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X(13)CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X(13)CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X(13)CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X(13)CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X(13)CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)(13)C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-(13)C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X(13)CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X(13)CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php .

High-repetition-rate three-dimensional OH imaging using scanned planar laser-induced fluorescence system for multiphase combustion.

Imaging dynamic multiphase combusting events is challenging. Conventional techniques can image only a single plane of an event, capturing limited details. Here, we report on a three-dimensional, time-resolved, OH planar laser-induced fluorescence (3D OH PLIF) technique that was developed to measure the relative OH concentration in multiphase combustion flow fields. To the best of our knowledge, this is the first time a 3D OH PLIF technique has been reported in the open literature. The technique involves rapidly scanning a laser sheet across a flow field of interest. The overall experimental system consists of a 5 kHz OH PLIF system, a high-speed detection system (image intensifier and CMOS camera), and a galvanometric scanning mirror. The scanning mirror was synchronized with a 500 Hz triangular sweep pattern generated using Labview. Images were acquired at 5 kHz corresponding to six images per mirror scan, and 1000 scans per second. The six images obtained in a scan were reconstructed into a volumetric representation. The resulting spatial resolution was 500×500×6 voxels mapped to a field of interest covering 30 mm×30 mm×8 mm. The novel 3D OH PLIF system was applied toward imaging droplet combustion of methanol gelled with hydroxypropyl cellulose (HPC) (3 wt. %, 6 wt. %), as well as solid propellant combustion, and impinging jet spray combustion. The resulting 3D dataset shows a comprehensive view of jetting events in gelled droplet combustion that was not observed with high-speed imaging or 2D OH PLIF. Although the scan is noninstantaneous, the temporal and spatial resolution was sufficient to view the dynamic events in the multiphase combustion flow fields of interest. The system is limited by the repetition rate of the pulsed laser and the step response time of the galvanometric mirror; however, the repetition rates are sufficient to resolve events in the order of 100 Hz. Future upgrade includes 40 kHz pulsed UV laser system, which can reduce the scan time to 125 µs, while keeping the high repetition rate of 1000 Hz.

Novel miRNA-inducing drugs enable differentiation of retinoic acid-resistant neuroblastoma cells.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and miR-132-inducing bufalin synergistically arrests proliferation, induces differentiation, and prolongs the survival of differentiated MES SK-N-AS cells for 8 weeks. RNA- seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained other MES neuroblastoma, as well as glioblastoma cells, over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.

Loss of mitochondrial pyruvate transport initiates cardiac glycogen accumulation and heart failure.

Background: Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. Methods: We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of U-13C-glucose. Cardiac glycogen was assessed biochemically and by transmission electron microscopy. Cardiac uptake of 2-deoxyglucose was measured and western blotting performed to analyze insulin signaling and enzymatic regulators of glycogen synthesis and degradation. Isotope tracing and glycogen analysis was also performed in hearts from mice fed either low-fat diet or a ketogenic diet previously shown to reverse the CS-Mpc2-/- heart failure. Cardiac glycogen was also assessed in mice infused with angiotensin-II that were fed low-fat or ketogenic diet. Results: Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, yet these hearts displayed increased enrichment from U-13C-glucose and increased glycolytic metabolite pool sizes. 13C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. This increased glycogen synthesis occurred despite enhanced inhibitory phosphorylation of glycogen synthase and reduced expression of glycogenin-1. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic 13C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin-II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Conclusions: Our results indicate that loss of MPC in the heart causes glycogen accumulation and heart failure, while a ketogenic diet can reverse both the glycogen accumulation and heart failure. We conclude that maintaining mitochondrial pyruvate import and metabolism is critical for the heart, unless cardiac pyruvate metabolism is reduced by consumption of a ketogenic diet.

Loss of dihydroceramide desaturase drives neurodegeneration by disrupting endoplasmic reticulum and lipid droplet homeostasis in glial cells.

Dihydroceramide desaturases convert dihydroceramides to ceramides, the precursors of all complex sphingolipids. Reduction of DEGS1 dihydroceramide desaturase function causes pediatric neurodegenerative disorder hypomyelinating leukodystrophy-18 (HLD-18). We discovered that infertile crescent (ifc), the Drosophila DEGS1 homolog, is expressed primarily in glial cells to promote CNS development by guarding against neurodegeneration. Loss of ifc causes massive dihydroceramide accumulation and severe morphological defects in cortex glia, including endoplasmic reticulum (ER) expansion, failure of neuronal ensheathment, and lipid droplet depletion. RNAi knockdown of the upstream ceramide synthase schlank in glia of ifc mutants rescues ER expansion, suggesting dihydroceramide accumulation in the ER drives this phenotype. RNAi knockdown of ifc in glia but not neurons drives neuronal cell death, suggesting that ifc function in glia promotes neuronal survival. Our work identifies glia as the primary site of disease progression in HLD-18 and may inform on juvenile forms of ALS, which also feature elevated dihydroceramide levels.

Inducing vulnerability to InhA inhibition restores isoniazid susceptibility in drug-resistant Mycobacterium tuberculosis.

Of the approximately 10 million cases of Mycobacterium tuberculosis (Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates the conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor of Mtb respiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for the growth of Mtb in high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of the Mtb electron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causing Mtb to become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability in Mtb that can be exploited to render Mtb sensitive to otherwise subinhibitory concentrations of InhA inhibitor.IMPORTANCEIsoniazid (INH) is a critical frontline antibiotic to treat Mycobacterium tuberculosis (Mtb) infections. INH efficacy is limited by its suboptimal penetration of the Mtb-containing lesion and by the prevalence of clinical INH resistance. We previously discovered a compound, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a set of INH-resistant mutants to INH. Resistance is typically mediated by katG mutations that decrease the activation of INH, which is required for INH to inhibit the essential enzyme InhA. Our current work demonstrates that C10 re-sensitizes INH-resistant katG-hypomorphs without enhancing the activation of INH. We furthermore show that C10 causes Mtb to become particularly vulnerable to InhA inhibition without compromising InhA activity on its own. Therefore, C10 represents a novel strategy to curtail the development of INH resistance and to sensitize Mtb to sub-lethal doses of INH, such as those achieved at the infection site.

Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.

Cardiometabolic characteristics of people with metabolically healthy and unhealthy obesity.

There is considerable heterogeneity in the cardiometabolic abnormalities associated with obesity. We evaluated multi-organ system metabolic function in 20 adults with metabolically healthy obesity (MHO; normal fasting glucose and triglycerides, oral glucose tolerance, intrahepatic triglyceride content, and whole-body insulin sensitivity), 20 adults with metabolically unhealthy obesity (MUO; prediabetes, hepatic steatosis, and whole-body insulin resistance), and 15 adults who were metabolically healthy lean. Compared with MUO, people with MHO had (1) altered skeletal muscle biology (decreased ceramide content and increased expression of genes involved in BCAA catabolism and mitochondrial structure/function); (2) altered adipose tissue biology (decreased expression of genes involved in inflammation and extracellular matrix remodeling and increased expression of genes involved in lipogenesis); (3) lower 24-h plasma glucose, insulin, non-esterified fatty acids, and triglycerides; (4) higher plasma adiponectin and lower plasma PAI-1 concentrations; and (5) decreased oxidative stress. These findings provide a framework of potential mechanisms responsible for MHO and the metabolic heterogeneity of obesity. This study was registered at ClinicalTrials.gov (NCT02706262).

A view from above: cloud plots to visualize global metabolomic data.

Global metabolomics describes the comprehensive analysis of small molecules in a biological system without bias. With mass spectrometry-based methods, global metabolomic data sets typically comprise thousands of peaks, each of which is associated with a mass-to-charge ratio, retention time, fold change, p-value, and relative intensity. Although several visualization schemes have been used for metabolomic data, most commonly used representations exclude important data dimensions and therefore limit interpretation of global data sets. Given that metabolite identification through tandem mass spectrometry data acquisition is a time-limiting step of the untargeted metabolomic workflow, simultaneous visualization of these parameters from large sets of data could facilitate compound identification and data interpretation. Here, we present such a visualization scheme of global metabolomic data using a so-called "cloud plot" to represent multidimensional data from septic mice. While much attention has been dedicated to lipid compounds as potential biomarkers for sepsis, the cloud plot shows that alterations in hydrophilic metabolites may provide an early signature of the disease prior to the onset of clinical symptoms. The cloud plot is an effective representation of global mass spectrometry-based metabolomic data, and we describe how to extract it as standard output from our XCMS metabolomic software.

Inducing vulnerability to InhA inhibition restores isoniazid susceptibility in drug resistant Mycobacterium tuberculosis.

Of the approximately 10 million cases of Mycobacterium tuberculosis (Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor of Mtb respiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for growth of Mtb in high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of the Mtb electron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causing Mtb to become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability in Mtb that can be exploited to render Mtb sensitive to otherwise subinhibitory concentrations of InhA inhibitor.

Effects of hepatic mitochondrial pyruvate carrier deficiency on de novo lipogenesis and glycerol-mediated gluconeogenesis in mice.

The liver coordinates the systemic response to nutrient deprivation and availability by producing glucose from gluconeogenesis during fasting and synthesizing lipids via de novo lipogenesis (DNL) when carbohydrates are abundant. Mitochondrial pyruvate metabolism is thought to play important roles in both gluconeogenesis and DNL. We examined the effects of hepatocyte-specific mitochondrial pyruvate carrier (MPC) deletion on the fasting-refeeding response. Rates of DNL during refeeding were impaired by liver MPC deletion, but this did not reduce intrahepatic lipid content. During fasting, glycerol is converted to glucose by two pathways; a direct cytosolic pathway essentially reversing glycolysis and an indirect mitochondrial pathway requiring the MPC. MPC deletion reduced the incorporation of 13C-glycerol into TCA cycle metabolites but not into newly synthesized glucose. However, suppression of glycerol metabolism did not affect glucose concentrations in fasted hepatocyte-specific MPC-deficient mice. Thus, glucose production by kidney and intestine may compensate for MPC deficiency in hepatocytes.

Mitochondrial Pyruvate Carrier Inhibition Attenuates Hepatic Stellate Cell Activation and Liver Injury in a Mouse Model of Metabolic Dysfunction-associated Steatotic Liver Disease.

Hepatic stellate cells (HSC) are non-parenchymal liver cells that produce extracellular matrix comprising fibrotic lesions in chronic liver diseases. Prior work demonstrated that mitochondrial pyruvate carrier (MPC) inhibitors suppress HSC activation and fibrosis in a mouse model of metabolic dysfunction-associated steatohepatitis (MASH). In the present study, pharmacologic or genetic inhibition of the MPC in HSC decreased expression of markers of activation in vitro. MPC knockdown also reduced the abundance of several intermediates of the TCA cycle, and diminished α-ketoglutarate played a key role in attenuating HSC activation by suppressing hypoxia inducible factor-1α signaling. On high fat diets, mice with HSC-specific MPC deletion exhibited reduced circulating transaminases, numbers of HSC, and hepatic expression of markers of HSC activation and inflammation compared to wild-type mice. These data suggest that MPC inhibition modulates HSC metabolism to attenuate activation and illuminate mechanisms by which MPC inhibitors could prove therapeutically beneficial for treating MASH.