Pyruvate induces torpor in obese mice.

Mice subjected to cold or caloric deprivation can reduce body temperature and metabolic rate and enter a state of torpor. Here we show that administration of pyruvate, an energy-rich metabolic intermediate, can induce torpor in mice with diet-induced or genetic obesity. This is associated with marked hypothermia, decreased activity, and decreased metabolic rate. The drop in body temperature correlates with the degree of obesity and is blunted by housing mice at thermoneutrality. Induction of torpor by pyruvate in obese mice relies on adenosine signaling and is accompanied by changes in brain levels of hexose bisphosphate and GABA as detected by mass spectroscopy-based imaging. Pyruvate does not induce torpor in lean mice but results in the activation of brown adipose tissue (BAT) with an increase in the level of uncoupling protein-1 (UCP1). Denervation of BAT in lean mice blocks this increase in UCP1 and allows the pyruvate-induced torpor phenotype. Thus, pyruvate administration induces torpor in obese mice by pathways involving adenosine and GABA signaling and a failure of normal activation of BAT.

Integrated, High-Throughput, Multiomics Platform Enables Data-Driven Construction of Cellular Responses and Reveals Global Drug Mechanisms of Action.

An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multiomics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10 000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resource to understand complex cellular responses in a high-throughput manner.

Identification of dimethyldioctadecylammonium ion (m/z 550.6) and related species (m/z 522.6, 494.6) as a source of contamination in mass spectrometry.

Chemical contamination can be one of the more common problems encountered when performing trace-level analysis regardless of the analytical technique. Minimizing or eliminating background interferences can be a difficult task, so knowledge of the chemical composition of these contaminants can prove invaluable when it comes to identifying the source. Once the source is identified, proper steps may be taken to reduce or eliminate it. In this study, we report the identity of some commonly seen contaminants (m/z 550.6, 522.6, and 494.6) in electrospray ionization (ESI) mass spectrometry (MS). Through MS, tandem MS, accurate-mass, and high-resolution measurements we have identified these background contaminants as being quaternary ammonium species that contain long-chain hydrocarbon groups, where m/z 550.6 is a dimethyldioctadecylammonium ion (C(18), C(18)) and m/z 522.6 and 494.6 are similar in nature but have shorter alkyl-chain groups. The lipophilic nature of these compounds and the fact that they have molecular weights similar to lysophospholipids make them a frequent contaminant in lipidomic studies. The likely sources of these compounds are commonly used personal and household products.

Regional differences in brain glucose metabolism determined by imaging mass spectrometry.

OBJECTIVE: Glucose is the major energy substrate of the brain and crucial for normal brain function. In diabetes, the brain is subject to episodes of hypo- and hyperglycemia resulting in acute outcomes ranging from confusion to seizures, while chronic metabolic dysregulation puts patients at increased risk for depression and Alzheimer's disease. In the present study, we aimed to determine how glucose is metabolized in different regions of the brain using imaging mass spectrometry (IMS). METHODS: To examine the relative abundance of glucose and other metabolites in the brain, mouse brain sections were subjected to imaging mass spectrometry at a resolution of 100 µm. This was correlated with immunohistochemistry, qPCR, western blotting and enzyme assays of dissected brain regions to determine the relative contributions of the glycolytic and pentose phosphate pathways to regional glucose metabolism. RESULTS: In brain, there are significant regional differences in glucose metabolism, with low levels of hexose bisphosphate (a glycolytic intermediate) and high levels of the pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolite hexose phosphate in thalamus compared to cortex. The ratio of ATP to ADP is significantly higher in white matter tracts, such as corpus callosum, compared to less myelinated areas. While the brain is able to maintain normal ratios of hexose phosphate, hexose bisphosphate, ATP, and ADP during fasting, fasting causes a large increase in cortical and hippocampal lactate. CONCLUSION: These data demonstrate the importance of direct measurement of metabolic intermediates to determine regional differences in brain glucose metabolism and illustrate the strength of imaging mass spectrometry for investigating the impact of changing metabolic states on brain function at a regional level with high resolution.

Blockade of nucleoside transport is required for delivery of intraarterial adenosine into the interstitium: relevance to therapeutic preconditioning in humans.

BACKGROUND: Adenosine, a known mediator of preconditioning, has been infused into the coronary circulation to induce therapeutic preconditioning, eg, in preparation for angioplasty. However, results have been disappointing. We tested the hypothesis that endothelial nucleoside transporter acts as a barrier impeding the delivery of intravascular adenosine into the underlying myocardium and that this can be overcome with dipyridamole, a nucleoside transporter blocker. METHODS AND RESULTS: We infused saline or adenosine (0.125 and 0.5 mg/min) into the brachial artery while monitoring forearm blood flow (FBF) and interstitial adenosine levels with microdialysis probes implanted in the flexor digitorum superficialis of the forearm in 7 healthy volunteers during intravenous administration of saline or dipyridamole (loading dose, 0.142 mg/kg per min for 5 minutes followed by 0.004 mg/kg per min). Adenosine produced near maximal forearm vasodilation, increasing FBF from 4.0+/-0.7 to 10.4+/-1.9 and 13.1+/-1.6 mL/100 mL per min for the low and high doses, respectively, but did not increase muscle dialysate adenosine concentration (from 88+/-21 to 65+/-23 and 85+/-26 nmol/L). Intravenous dipyridamole enhanced resting muscle dialysate adenosine (from 77+/-25 to 147+/-50 nmol/L), adenosine-induced increase in FBF (from 4.1+/-0.8 to 12.6+/-3 and 15.1+/-3 mL/100 mL per min for the low and high dose, respectively), and the delivery of adenosine into the interstitium (to 290+/-80 and 299+/-143 nmol/L for the low and high dose, respectively, P=0.04). CONCLUSIONS: Intravascular adenosine is likely ineffective in inducing myocardial preconditioning because of poor interstitial delivery. This can be overcome by blocking the nucleoside transporter with dipyridamole.

Biosynthetic origins of C-P bond containing tripeptide K-26.

[reaction: see text] Primary metabolic precursors for K-26, a naturally occurring tripeptide phosphonic acid from Actinomyces sp. K-26, are investigated by heavy-atom isotope labeled substrate incorporation experiments. A highly sensitive selected reaction monitoring (SRM)-based method for isotopic incorporation estimation in natural products is reported. The incorporation of heavy-atom isotope labeled tyrosine compounds into the (R)-1-amino-2-(4-hydroxyphenyl)-ethylphosphonic acid moiety of compound K-26 suggests a new mechanism of biosynthesis of phosphonate functionality in natural products.

Detection of the 15-acetate of prostaglandin E2 methyl ester as a prominent component of the prostaglandins in the gorgonian coral Plexaura homomalla.

15R-Prostaglandin E2 (PGE2) methyl ester 15-acetate (1) was isolated from the R-variety of the Caribbean sea whip coral Plexaura homomalla collected in the Florida Keys. It was present in coral samples from separate collections in 2-10% of the abundance of the major prostaglandin component, PGA2 methyl ester 15-acetate. The structure of 1 was assigned based on one- and two-dimensional 1H NMR, HPLC, and LC-MS analyses. A sample of the S-variety of P. homomalla was found to contain a similar abundance of the corresponding 15S product, prostaglandin E2 methyl ester 15-acetate. The significance of PGE acetylation is discussed in relation to the proposed mechanism of PGA synthesis in the coral.

A derivatization and validation strategy for determining the spatial localization of endogenous amine metabolites in tissues using MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here, we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high-mass resolution and MS(n) IMS. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high-performance liquid chromatography (HPLC)-MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds.

Monitoring the inflammatory response to infection through the integration of MALDI IMS and MRI.

Systemic bacterial infection is characterized by a robust whole-organism inflammatory response. Analysis of the immune response to infection involves technologies that typically focus on single organ systems and lack spatial information. Additionally, the analysis of individual inflammatory proteins requires antibodies specific to the protein of interest, limiting the panel of proteins that can be analyzed. Herein we describe the application of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) to mice systemically infected with Staphylococcus aureus to identify inflammatory protein masses that respond to infection throughout an entire infected animal. Integrating the resolution afforded by magnetic resonance imaging (MRI) with the sensitivity of MALDI IMS provides three-dimensional spatially resolved information regarding the distribution of innate immune proteins during systemic infection, allowing comparisons to in vivo structural information and soft-tissue contrast via MRI. Thus, integrating MALDI IMS with MRI provides a systems-biology approach to study inflammation during infection.

Reagent precoated targets for rapid in-tissue derivatization of the anti-tuberculosis drug isoniazid followed by MALDI imaging mass spectrometry.

Isoniazid (INH) is an important component of front-line anti-tuberculosis therapy with good serum pharmacokinetics but unknown ability to penetrate tuberculous lesions. However, endogenous background interferences hinder our ability to directly analyze INH in tissues. Chemical derivatization has been successfully used to measure isoniazid directly from tissue samples using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). MALDI targets were pretreated with trans-cinnamaldehyde (CA) prior to mounting tissue slices. Isoniazid present in the tissues was efficiently derivatized and the INH-CA product measured by MS/MS. Precoating of MALDI targets allows the tissues to be directly thaw-mounted and derivatized, thus simplifying the preparation. A time-course series of tissues from tuberculosis infected/INH dosed animals were assayed and the MALDI MS/MS response correlates well with the amount of INH determined to be in the tissues by high-performance liquid chromatography (HPLC)-MS/MS.

Lung cancer diagnosis from proteomic analysis of preinvasive lesions.

Early detection may help improve survival from lung cancer. In this study, our goal was to derive and validate a signature from the proteomic analysis of bronchial lesions that could predict the diagnosis of lung cancer. Using previously published studies of bronchial tissues, we selected a signature of nine matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) mass-to-charge ratio features to build a prediction model diagnostic of lung cancer. The model was based on MALDI MS signal intensity (MALDI score) from bronchial tissue specimens from our 2005 published cohort of 51 patients. The performance of the prediction model in identifying lung cancer was tested in an independent cohort of bronchial specimens from 60 patients. The probability of having lung cancer based on the proteomic analysis of the bronchial specimens was characterized by an area under the receiver operating characteristic curve of 0.77 (95% CI 0.66-0.88) in this validation cohort. Eight of the nine features were identified and validated by Western blotting and immunohistochemistry. These results show that proteomic analysis of endobronchial lesions may facilitate the diagnosis of lung cancer and the monitoring of high-risk individuals for lung cancer in surveillance and chemoprevention trials.