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Abstract Light is fundamental for biological life, with most mammals possessing light-sensing photoreceptors in various organs. Opsin3 is highly expressed in adipose tissue which has extensive communication with other organs, particularly with the brain through the sympathetic nervous system (SNS). Our study reveals a new light-triggered crosstalk between adipose tissue and the hypothalamus. Direct blue-light exposure to subcutaneous white fat improves high-fat diet-induced metabolic abnormalities in an Opsin3-dependent manner. Metabolomic analysis shows that blue light increases circulating levels of histidine, which activates histaminergic neurons in the hypothalamus and stimulates brown adipose tissue (BAT) via SNS. Blocking central actions of histidine and denervating peripheral BAT blunts the effects of blue light. Human white adipocytes respond to direct blue light stimulation in a cell-autonomous manner, highlighting the translational relevance of this pathway. Together, these data demonstrate a light-responsive metabolic circuit involving adipose-hypothalamus communication, offering a potential strategy to alleviate obesity-induced metabolic abnormalities.

Abstract Activating brown adipose tissue (BAT) improves systemic metabolism, making it a promising target for metabolic syndrome. BAT is activated by 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), which we previously identified to be inversely associated with BMI and which directly improves metabolism in multiple tissues. Here we profile plasma lipidomics from 83 people and test which lipids’ association with BMI replicates in a concordant direction using our novel tool ScreenDMT, whose power and validity we demonstrate via mathematical proofs and simulations. We find that the linoleic acid diols 12,13-diHOME and 9,10-diHOME are both replicably inversely associated with BMI and mechanistically activate calcium influx in mouse brown and white adipocytes in vitro, which implicates this signaling pathway and 9,10-diHOME as candidate therapeutic targets. ScreenDMT can be applied to test directional mediation, directional replication, and qualitative interactions, such as identifying biomarkers whose association is shared (replication) or opposite (qualitative interaction) across diverse populations.

Abstract Parkinson’s disease is a progressive neurodegenerative disorder in which loss of dopaminergic neurons in the substantia nigra results in a clinically heterogeneous group with variable motor and non-motor symptoms with a degree of misdiagnosis. Only 3–25% of sporadic Parkinson’s patients present with genetic abnormalities that could represent a risk factor, thus environmental, metabolic, and other unknown causes contribute to the pathogenesis of Parkinson’s disease, which highlights the critical need for biomarkers. In the present study, we prospectively collected and analyzed plasma samples from 194 Parkinson’s disease patients and 197 age-matched non-diseased controls. N-acetyl putrescine (NAP) in combination with sense of smell (B-SIT), depression/anxiety (HADS), and acting out dreams (RBD1Q) clinical measurements demonstrated combined diagnostic utility. NAP was increased by 28% in Parkinsons disease patients and exhibited an AUC of 0.72 as well as an OR of 4.79. The clinical and NAP panel demonstrated an area under the curve, AUC = 0.9 and an OR of 20.4. The assessed diagnostic panel demonstrates combinatorial utility in diagnosing Parkinson’s disease, allowing for an integrated interpretation of disease pathophysiology and highlighting the use of multi-tiered panels in neurological disease diagnosis.

Abstract Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

Summary: Exercise mediates tissue metabolic function through direct and indirect adaptations to acylcarnitine (AC) metabolism, but the exact mechanisms are unclear. We found that circulating medium-chain acylcarnitines (AC) (C12-C16) are lower in active/endurance trained human subjects compared to sedentary controls, and this is correlated with elevated cardiorespiratory fitness and reduced adiposity. In mice, exercise reduced serum AC and increased liver AC, and this was accompanied by a marked increase in expression of genes involved in hepatic AC metabolism and mitochondrial β-oxidation. Primary hepatocytes from high-fat fed, exercise trained mice had increased basal respiration compared to hepatocytes from high-fat fed sedentary mice, which may be attributed to increased Ca2+ cycling and lipid uptake into mitochondria. The addition of specific medium- and long-chain AC to sedentary hepatocytes increased mitochondrial respiration, mirroring the exercise phenotype. These data indicate that AC redistribution is an exercise-induced mechanism to improve hepatic function and metabolism.

Abstract Cancer biomarker discovery is critically dependent on the integrity of biofluid and tissue samples acquired from study participants. Multi-omic profiling of candidate protein, lipid, and metabolite biomarkers is confounded by timing and fasting status of sample collection, participant demographics and treatment exposures of the study population. Contamination by hemoglobin, whether caused by hemolysis during sample preparation or underlying red cell fragility, contributes 0–10 g/L of extraneous protein to plasma, serum, and Buffy coat samples and may interfere with biomarker detection and validation. We analyzed 617 plasma, 701 serum, and 657 buffy coat samples from a 7-year longitudinal multi-omic biomarker discovery program evaluating 400+ participants with or at risk for pancreatic cancer, known as Project Survival. Hemolysis was undetectable in 93.1% of plasma and 95.0% of serum samples, whereas only 37.1% of buffy coat samples were free of contamination by hemoglobin. Regression analysis of multi-omic data demonstrated a statistically significant correlation between hemoglobin concentration and the resulting pattern of analyte detection and concentration. Although hemolysis had the greatest impact on identification and quantitation of the proteome, distinct differentials in metabolomics and lipidomics were also observed and correlated with severity. We conclude that quality control is vital to accurate detection of informative molecular differentials using OMIC technologies and that caution must be exercised to minimize the impact of hemolysis as a factor driving false discovery in large cancer biomarker studies.

Abstract Prostate-specific antigen (PSA) screening for prostate cancer (PCa) is limited by the lack of specificity but is further complicated in the benign prostatic hyperplasia (BPH) population which also exhibit elevated PSA, representing a clear unmet need to distinguish BPH from PCa. Herein, we evaluated the utility of FLNA IP-MRM, age, and prostate volume to stratify men with BPH from those with PCa. Diagnostic performance of the biomarker panel was better than PSA alone in discriminating patients with negative biopsy from those with PCa, as well as those who have had multiple prior biopsies (AUC 0.75 and 0.87 compared to AUC of PSA alone 0.55 and 0.57 for patients who have had single compared to multiple negative biopsies, respectively). Of interest, in patients with PCa, the panel demonstrated improved performance than PSA alone in those with Gleason scores of 5–7 (AUC 0.76 vs. 0.56) and Gleason scores of 8–10 (AUC 0.74 vs. 0.47). With Gleason scores (8–10), the negative predictive value of the panel is 0.97, indicating potential to limit false negatives in aggressive cancers. Together, these data demonstrate the ability of the biomarker panel to perform better than PSA alone in men with BPH, thus preventing unnecessary biopsies.

Abstract Reactive oxygen species (ROS) are implicated in triggering cell signalling events and pathways to promote and maintain tumorigenicity. Chemotherapy and radiation can induce ROS to elicit cell death allows for targeting ROS pathways for effective anti-cancer therapeutics. Coenzyme Q10 is a critical cofactor in the electron transport chain with complex biological functions that extend beyond mitochondrial respiration. This study demonstrates that delivery of oxidized Coenzyme Q10 (ubidecarenone) to increase mitochondrial Q-pool is associated with an increase in ROS generation, effectuating anti-cancer effects in a pancreatic cancer model. Consequent activation of cell death was observed in vitro in pancreatic cancer cells, and both human patient-derived organoids and tumour xenografts. The study is a first to demonstrate the effectiveness of oxidized ubidecarenone in targeting mitochondrial function resulting in an anti-cancer effect. Furthermore, these findings support the clinical development of proprietary formulation, BPM31510, for treatment of cancers with high ROS burden with potential sensitivity to ubidecarenone.

Abstract Metabolic reprogramming in cancer cells, vs. non-cancer cells, elevates levels of reactive oxygen species (ROS) leading to higher oxidative stress. The elevated ROS levels suggest a vulnerability to excess prooxidant loads leading to selective cell death, a therapeutically exploitable difference. Co-enzyme Q10 (CoQ10) an endogenous mitochondrial resident molecule, plays an important role in mitochondrial redox homeostasis, membrane integrity, and energy production. BPM31510 is a lipid-drug conjugate nanodispersion specifically formulated for delivery of supraphysiological concentrations of ubidecarenone (oxidized CoQ10) to the cell and mitochondria, in both in vitro and in vivo model systems. In this study, we sought to investigate the therapeutic potential of ubidecarenone in the highly treatment-refractory glioblastoma. Rodent (C6) and human (U251) glioma cell lines, and non-tumor human astrocytes (HA) and rodent NIH3T3 fibroblast cell lines were utilized for experiments. Tumor cell lines exhibited a marked increase in sensitivity to ubidecarenone vs. non-tumor cell lines. Further, elevated mitochondrial superoxide production was noted in tumor cells vs. non-tumor cells hours before any changes in proliferation or the cell cycle could be detected. In vitro co-culture experiments show ubidecarenone differentially affecting tumor cells vs. non-tumor cells, resulting in an equilibrated culture. In vivo activity in a highly aggressive orthotopic C6 glioma model demonstrated a greater than 25% long-term survival rate. Based on these findings we conclude that high levels of ubidecarenone delivered using BPM31510 provide an effective therapeutic modality targeting cancer-specific modulation of redox mechanisms for anti-cancer effects.

Abstract Androgen deprivation therapy (ADT) is the main treatment strategy for men with metastatic prostate cancer (PC). However, ADT is associated with various metabolic disturbances, including impaired glucose tolerance, insulin resistance and weight gain, increasing risk of diabetes and cardiovascular death. Much remains unknown about the metabolic pathways and disturbances altered by ADT and the mechanisms. We assessed the metabolomic effects of ADT in the serum of 20 men receiving ADT. Sera collected before (baseline), 3 and 6 months after initiation of ADT was used for the metabolomics and lipidomics analyses. The ADT‐associated metabolic changes were identified by univariable and multivariable statistical analysis, ANOVA, and Pearson correlation. We found multiple key changes. First, ADT treatments reduced the steroid synthesis as reflected by the lower androgen sulfate and other steroid hormones. Greater androgen reduction was correlated with higher serum glucose levels, supporting the diabetogenic role of ADT. Second, ADT consistently decreased the 3‐hydroxybutyric acid and ketogenesis. Third, many acyl‐carnitines were reduced, indicating the effects on the fatty acid metabolism. Fourth, ADT was associated with a corresponding reduction in 3‐formyl indole (a.k.a. indole‐3‐carboxaldehyde), a microbiota‐derived metabolite from the dietary tryptophan. Indole‐3‐carboxaldehyde is an agonist for the aryl hydrocarbon receptor and regulates the mucosal reactivity and inflammation. Together, these ADT‐associated metabolomic analyses identified reduction in steroid synthesis and ketogenesis as prominent features, suggesting therapeutic potential of restricted ketogenic diets, though this requires formal testing. ADT may also impact the microbial production of indoles related to the immune pathways. Future research is needed to determine the functional impact and underlying mechanisms to prevent ADT‐linked comorbidities and diabetes risk.

AbstractThe spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase–anaphase transition. Here, we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae

Abstract Background Predicting the clinical course of prostate cancer is challenging due to the wide biological spectrum of the disease. The objective of our study was to identify prostate cancer prognostic markers in patients ‘sera using a multi-omics discovery platform. Methods Pre-surgical serum samples collected from a longitudinal, racially diverse, prostate cancer patient cohort (N = 382) were examined. Linear Regression and Bayesian computational approaches integrated with multi-omics, were used to select markers to predict biochemical recurrence (BCR). BCR-free survival was modeled using unadjusted Kaplan–Meier estimation curves and multivariable Cox proportional hazards analysis, adjusted for key pathologic variables. Receiver operating characteristic (ROC) curve statistics were used to examine the predictive value of markers in discriminating BCR events from non-events. The findings were further validated by creating a training set (N = 267) and testing set (N = 115) from the cohort. Results Among 382 patients, 72 (19%) experienced a BCR event in a median follow-up time of 6.9 years. Two proteins—Tenascin C (TNC) and Apolipoprotein A1V (Apo-AIV), one metabolite—1-Methyladenosine (1-MA) and one phospholipid molecular species phosphatidic acid (PA) 18:0-22:0 showed a cumulative predictive performance of AUC = 0.78 [OR (95% CI) = 6.56 (2.98–14.40), P 80%) for BCR. The combination of pTstage and Gleason score with the analytes, further increased the sensitivity [AUC = 0.89, 95% (CI) = 4.45–32.05, P

Summary: Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in serum-deprived conditions are enriched in six protein pathways and one lipid class, dilysocardiolipin. Inspired by these findings, we engineer an “artificial exosome,” in which the incorporation of one lipid (dilysocardiolipin) and three proteins (Rab7, Desmoplakin, and AHSG) into conventional neutral liposomes produces vesicles that mimic cargo delivering activity of natural exosomes. : Biochemistry; Biological Sciences; Lipidomics; Molecular Biology; Proteomics Subject Areas: Biochemistry, Biological Sciences, Lipidomics, Molecular Biology, Proteomics

Summary: Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.

Abstract Background Diabetes mellitus is associated with an increased risk in diabetic cardiomyopathy (DCM) that is distinctly not attributed to co-morbidities with other vasculature diseases. To date, while dysregulation of calcium handling is a key hallmark in cardiomyopathy, studies have been inconsistent in the types of alterations involved. In this study human cardiomyocytes were exposed to an environmental nutritional perturbation of high glucose, fatty acids, and l-carnitine to model DCM and iTRAQ-coupled LC–MS/MS proteomic analysis was used to capture proteins affected by the perturbation. The proteins captured were then compared to proteins currently annotated in the cardiovascular disease (CVD) gene ontology (GO) database to identify proteins not previously described as being related to CVD. Subsequently, GO analysis for calcium regulating proteins and endoplasmic/sarcoplasmic reticulum (ER/SR) associated proteins was carried out. Results Here, we identified CCDC47 (calumin) as a unique calcium regulating protein altered in our in vitro nutritional perturbation model. The cellular and functional role of CCDC47 was then assessed in rat cardiomyocytes. In rat H9C2 myocytes, overexpression of CCDC47 resulted in increase in ionomycin-induced calcium release and reuptake. Of interest, in a diet-induced obese (DIO) rat model of DCM, CCDC47 mRNA expression was increased in the atrium and ventricle of the heart, but CCDC47 protein expression was significantly increased only in the atrium of DIO rats compared to lean control rats. Notably, no changes in ANP, BNP, or β-MHC were observed between DIO rats and lean control rats. Conclusions Together, our in vitro and in vivo studies demonstrate that CCDC47 is a unique calcium regulating protein that is associated with early onset hypertrophic cardiomyopathy.

Summary: Thermogenic fat expends energy during cold for temperature homeostasis, and its activity regulates nutrient metabolism and insulin sensitivity. We measured cold-activated lipid landscapes in circulation and in adipose tissue by MS/MSALL shotgun lipidomics. We created an interactive online viewer to visualize the changes of specific lipid species in response to cold. In adipose tissue, among the approximately 1,600 lipid species profiled, we identified the biosynthetic pathway of the mitochondrial phospholipid cardiolipin as coordinately activated in brown and beige fat by cold in wild-type and transgenic mice with enhanced browning of white fat. Together, these data provide a comprehensive lipid bio-signature of thermogenic fat activation in circulation and tissue and suggest pathways regulated by cold exposure. : In this resource, Lynes et al. use a sensitive MS/MSALL technique to profile over 1,000 lipid species in the blood and adipose tissue of humans and mice exposed to cold. Using the online viewer they created to view these data, pathways activated by cold exposure can be identified. Keywords: brown adipose tissue, white adipose tissue, lipidomics, thermogenesis, cardiolipin

Abstract Background Inhibition of Hsp90 has been shown to improve glucose tolerance and insulin sensitivity in mouse models of diabetes. In the present report, the specific isoform Hsp90ab1, was identified as playing a major role in regulating insulin signaling and glucose metabolism. Methods In a diet-induced obese (DIO) mouse model of diabetes, expression of various Hsp90 isoforms in skeletal tissue was examined. Subsequent experiments characterized the role of Hsp90ab1 isoform in glucose metabolism and insulin signaling in primary human skeletal muscle myoblasts (HSMM) and a DIO mouse model. Results In DIO mice Hsp90ab1 mRNA was upregulated in skeletal muscle compared to lean mice and knockdown using anti-sense oligonucleotide (ASO) resulted in reduced expression in skeletal muscle that was associated with improved glucose tolerance, reduced fed glucose and fed insulin levels compared to DIO mice that were treated with a negative control oligonucleotide. In addition, knockdown of HSP90ab1 in DIO mice was associated with reduced pyruvate dehydrogenase kinase-4 mRNA and phosphorylation of the muscle pyruvate dehydrogenase complex (at serine 232, 293 and 300), but increased phosphofructokinase 1, glycogen synthase 1 and long-chain specific acyl-CoA dehydrogenase mRNA. In HSMM, siRNA knockdown of Hsp90ab1 induced an increase in substrate metabolism, mitochondrial respiration capacity, and insulin sensitivity, providing further evidence for the role of Hsp90ab1 in metabolism. Conclusions The data support a novel role for Hsp90ab1 in arbitrating skeletal muscle plasticity via modulation of substrate utilization including glucose and fatty acids in normal and disease conditions. Hsp90ab1 represents a novel target for potential treatment of metabolic disease including diabetes.

Abstract Background Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. Results In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the “sequence varying mRNAs” transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. Conclusions Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well.

The emergence of COVID-19 progressed into a global pandemic that has functionally put the world at a standstill and catapulted major healthcare systems into an overburdened state. The dire need for therapeutic strategies to mitigate and successfully treat COVID-19 is now a public health crisis with national security implications for many countries. The current study employed Bayesian networks to a longitudinal proteomic dataset generated from Caco-2 cells transfected with SARS-CoV-2 (isolated from patients returning from Wuhan to Frankfurt). Two different approaches were employed to assess the Bayesian models, a titer-center topology analysis and a drug signature enrichment analysis. Topology analysis identified a set of proteins directly linked to the SAR-CoV2 titer, including ACE2, a SARS-CoV-2 binding receptor, MAOB and CHECK1. Aligning with the topology analysis, MAOB and CHECK1 were also identified within the enriched drug-signatures. Taken together, the data output from this network has identified nodal host proteins that may be connected to 18 chemical compounds, some already marketed, which provides an immediate opportunity to rapidly triage these assets for safety and efficacy against COVID-19.

Exercise improves whole-body metabolic health through adaptations to various tissues, including adipose tissue, but the effects of exercise training on the lipidome of white adipose tissue (WAT) and brown adipose tissue (BAT) are unknown. Here, we utilize MS/MSALL shotgun lipidomics to determine the molecular signatures of exercise-induced adaptations to subcutaneous WAT (scWAT) and BAT. Three weeks of exercise training decrease specific molecular species of phosphatidic acid (PA), phosphatidylcholines (PC), phosphatidylethanolamines (PE), and phosphatidylserines (PS) in scWAT and increase specific molecular species of PC and PE in BAT. Exercise also decreases most triacylglycerols (TAGs) in scWAT and BAT. In summary, exercise-induced changes to the scWAT and BAT lipidome are highly specific to certain molecular lipid species, indicating that changes in tissue lipid content reflect selective remodeling in scWAT and BAT of both phospholipids and glycerol lipids in response to exercise training, thus providing a comprehensive resource for future studies of lipid metabolism pathways.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types.

Widespread application of omic technologies is evolving our understanding of population health and holds promise in providing precise guidance for selection of therapeutic interventions based on patient biology. The opportunity to use hundreds of analytes for diagnostic assessment of human health compared to the current use of 10–20 analytes will provide greater accuracy in deconstructing the complexity of human biology in disease states. Conventional biochemical measurements like cholesterol, creatinine, and urea nitrogen are currently used to assess health status; however, metabolomics captures a comprehensive set of analytes characterizing the human phenotype and its complex metabolic processes in real-time. Unlike conventional clinical analytes, metabolomic profiles are dramatically influenced by demographic and environmental factors that affect the range of normal values and increase the risk of false biomarker discovery. This review addresses the challenges and opportunities created by the evolving field of clinical metabolomics and highlights features of study design and bioinformatics necessary to maximize the utility of metabolomics data across demographic groups.

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1β, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity.

(1) Background: Interest in the application of metabolomics toward clinical diagnostics development and population health monitoring has grown significantly in recent years. In spite of several advances in analytical and computational tools, obtaining a sufficient number of samples from patients remains an obstacle. The dried blood spot (DBS) and dried urine strip (DUS) methodologies are a minimally invasive sample collection method allowing for the relative simplicity of sample collection and minimal cost. (2) Methods: In the current report, we compared results of targeted metabolomics analyses of four types of human blood sample collection methods (with and without DBS) and two types of urine sample collection (DUS and urine) across several parameters including the metabolite coverage of each matrix and the sample stability for DBS/DUS using commercially available Whatman 903TM paper. The DBS/DUS metabolomics protocols were further applied to examine the temporal metabolite level fluctuations within hours and days of sample collection. (3) Results: Several hundred polar metabolites were monitored using DBS/DUS. Temporal analysis of the polar metabolites at various times of the day and across days identified several species that fluctuate as a function of day and time. In addition, a subset of metabolites were identified to be significantly altered across hours within a day and within successive days of the week. (4) Conclusion: A comprehensive DBS/DUS metabolomics protocol was developed for human blood and urine analyses. The described methodology demonstrates the potential for enabling patients to contribute to the expanding bioanalytical demands of precision medicine and population health studies.

Monoacylglycerols (MAGs) are structural and bioactive metabolites critical for biological function. Development of facile tools for measuring MAG are essential to understand its role in different diseases and various pathways. A data-independent acquisition method, MS/MSALL, using electrospray ionization (ESI) coupled quadrupole time of flight mass spectrometry (MS), was utilized for the structural identification and quantitative analysis of individual MAG molecular species. Compared with other acylglycerols, diacylglycerols (DAG) and triacylglycerols (TAG), MAG characteristically presented as a dominant protonated ion, [M + H]+, and under low collision energy as fatty acid-like fragments due to the neutral loss of the glycerol head group. At low concentrations (

Activating brown adipose tissue (BAT) improves systemic metabolism, making it a promising target for metabolic syndrome. BAT is activated by 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), which we previously identified to be inversely associated with BMI and which directly improves metabolism in multiple tissues. Here we profile plasma lipidomics from 83 people and test which lipids' association with BMI replicates in a concordant direction using our novel tool ScreenDMT, whose power and validity we demonstrate via mathematical proofs and simulations. We find that the linoleic acid diols 12,13-diHOME and 9,10-diHOME are both replicably inversely associated with BMI and mechanistically activate calcium influx in mouse brown and white adipocytes in vitro, which implicates this signaling pathway and 9,10-diHOME as candidate therapeutic targets. ScreenDMT can be applied to test directional mediation, directional replication, and qualitative interactions, such as identifying biomarkers whose association is shared (replication) or opposite (qualitative interaction) across diverse populations. A study of blood lipids in two cohorts using a new replication test, ScreenDMT, finds that people with lower BMI have higher levels of linoleic acid diols 12,13-diHOME and 9,10-diHOME, which upregulate adipocyte calcium influx. [ABSTRACT FROM AUTHOR]

Light is fundamental for biological life, with most mammals possessing light-sensing photoreceptors in various organs. Opsin3 is highly expressed in adipose tissue which has extensive communication with other organs, particularly with the brain through the sympathetic nervous system (SNS). Our study reveals a new light-triggered crosstalk between adipose tissue and the hypothalamus. Direct blue-light exposure to subcutaneous white fat improves high-fat diet-induced metabolic abnormalities in an Opsin3-dependent manner. Metabolomic analysis shows that blue light increases circulating levels of histidine, which activates histaminergic neurons in the hypothalamus and stimulates brown adipose tissue (BAT) via SNS. Blocking central actions of histidine and denervating peripheral BAT blunts the effects of blue light. Human white adipocytes respond to direct blue light stimulation in a cell-autonomous manner, highlighting the translational relevance of this pathway. Together, these data demonstrate a light-responsive metabolic circuit involving adipose-hypothalamus communication, offering a potential strategy to alleviate obesity-induced metabolic abnormalities. Light is essential for biological life. Here, the authors show blue light activates the photoreceptor Opsin3 in white fat, triggering a light-responsive metabolic circuit involving adipose-hypothalamus communication, which could potentially alleviate obesity-induced metabolic abnormalities. [ABSTRACT FROM AUTHOR]