Arjun Sengupta

Oxalic acid and diacylglycerol 36:3 are cross-species markers of sleep debt

Significance Reduced sleep duration is a hallmark of modern-day society and is increasingly associated with medical conditions, such as diabetes, obesity, metabolic syndrome, and cardiovascular disease. Here we present data from a rat model and human clinical study of chronic sleep restriction, both revealing that two metabolites in blood, oxalic acid and diacylglycerol 36:3, are quantitatively depleted under sleep-restricted conditions and restored after recovery sleep. Our findings also reveal a significant overall shift in lipid metabolism, with higher levels of phospholipids in both species and evidence of a systemic oxidative environment. This work provides a potential link between the known pathologies of reduced sleep duration and metabolic dysfunction. Sleep is an essential biological process that is thought to have a critical role in metabolic regulation. In humans, reduced sleep duration has been associated with risk for metabolic disorders, including weight gain, diabetes, obesity, and cardiovascular disease. However, our understanding of the molecular mechanisms underlying effects of sleep loss is only in its nascent stages. In this study we used rat and human models to simulate modern-day conditions of restricted sleep and addressed cross-species consequences via comprehensive metabolite profiling. Serum from sleep-restricted rats was analyzed using polar and nonpolar methods in two independent datasets (n = 10 per study, 3,380 measured features, 407 identified). A total of 38 features were changed across independent experiments, with the majority classified as lipids (18 from 28 identified). In a parallel human study, 92 metabolites were identified as potentially significant, with the majority also classified as lipids (32 of 37 identified). Intriguingly, two metabolites, oxalic acid and diacylglycerol 36:3, were robustly and quantitatively reduced in both species following sleep restriction, and recovered to near baseline levels after sleep restriction (P < 0.05, false-discovery rate < 0.2). Elevated phospholipids were also noted after sleep restriction in both species, as well as metabolites associated with an oxidizing environment. In addition, polar metabolites reflective of neurotransmitters, vitamin B3, and gut metabolism were elevated in sleep-restricted humans. These results are consistent with induction of peroxisome proliferator-activated receptors and disruptions of the circadian clock. The findings provide a potential link between known pathologies of reduced sleep duration and metabolic dysfunction, and potential biomarkers for sleep loss.

Candida Albicans Stimulates Streptococcus Mutans Microcolony Development via Cross-Kingdom Biofilm-Derived Metabolites

Candida albicans is frequently detected with heavy infection of Streptococcus mutans in plaque-biofilms from children affected with early-childhood caries, a prevalent and costly oral disease. The presence of C. albicans enhances S. mutans growth within biofilms, yet the chemical interactions associated with bacterial accumulation remain unclear. Thus, this study was conducted to investigate how microbial products from this cross-kingdom association modulate S. mutans build-up in biofilms. Our data revealed that bacterial-fungal derived conditioned medium (BF-CM) significantly increased the growth of S. mutans and altered biofilm 3D-architecture in a dose-dependent manner, resulting in enlarged and densely packed bacterial cell-clusters (microcolonies). Intriguingly, BF-CM induced S. mutans gtfBC expression (responsible for Gtf exoenzymes production), enhancing Gtf activity essential for microcolony development. Using a recently developed nanoculture system, the data demonstrated simultaneous microcolony growth and gtfB activation in situ by BF-CM. Further metabolites/chromatographic analyses of BF-CM revealed elevated amounts of formate and the presence of Candida-derived farnesol, which is commonly known to exhibit antibacterial activity. Unexpectedly, at the levels detected (25–50 μM), farnesol enhanced S. mutans-biofilm cell growth, microcolony development, and Gtf activity akin to BF-CM bioactivity. Altogether, the data provide new insights on how extracellular microbial products from cross-kingdom interactions stimulate the accumulation of a bacterial pathogen within biofilms.

Host metabolic responses to Plasmodium falciparum infections evaluated by 1H NMR metabolomics

The human malarial parasite Plasmodium falciparum causes the most severe forms of malarial infections, which include cerebral malaria and various organ dysfunctions amongst adults in India. So far no dependable clinical descriptor is available that can distinguish cerebral malaria from other symptomatically similar diseases such as sepsis and encephalitis. This study aims at evaluating the differential metabolic features of plasma samples from P. falciparum patients with varying severities, and patients suffering from symptomatically similar diseases. 1H Nuclear Magnetic Resonance (NMR) based metabolic profiling of the plasma of the infected individuals and the control population was performed. The differences in the plasma profiles were evaluated through multivariate statistical analyses. The results suggest malaria-specific elevation of plasma lipoproteins. Such an increase was absent in control populations. In addition, cerebral malaria patients exhibited a decrease in plasma glycoproteins; such a reduction was not observed in malarial patients without cerebral symptoms. The data presented here indicates that the metabolism and/or transport of the plasma lipids is specifically perturbed by malarial infections. The differential perturbation of the plasma glycoprotein levels in cerebral malaria patients may have important implications in the diagnosis of cerebral malaria.

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis.

Background: Components of GABA catabolism feed into sleep and potential energy pathways. Results: We identified a metabolic phenotype in Drosophila mutants of GABA turnover and traced it to a limit in glutamate, which is not relevant for sleep. Conclusion: GABA regulates metabolic and sleep homeostasis through independent mechanisms. Significance: Neurological disorders involving GABA disruption may be associated with metabolic problems. Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD+/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.

Sleep restriction induced energy, methylation and lipogenesis metabolic switches in rat liver

Sleep curtailment is ubiquitous in modern day society. Sleep debt is associated with maladaptive physiological changes that can lead to cardiometabolic and neuropsychiatric pathologies. Recent literature has shown the effects of sleep restriction (SR) on systemic metabolic profiles in biofluids, implying that tissue-specific metabolomes are impacted by SR. To test this hypothesis, we assessed hepatic metabolic profiles of rats after 5days of SR using UPLC-MS based metabolomics analysis and gene expression analysis. Our data suggests distinctive effects of SR on the liver metabolic profile of rats compared to forced-activity control animals. We observed specific impacts of SR on NAD metabolism through NAD accumulation and upregulation of Nampt, the rate determining step of NAD salvage. Additional multi-omic changes were observed in methionine metabolism, with an elevated SAM:SAH ratio under SR. This effect on one carbon metabolism is indicative of increased methylation potential. Changes in TCA cycle intermediates and ATP-citrate lyase (Acly) gene expression were observed that may be related to altered circulatory lipid profiles previously reported documenting the chrono-metabolic connection. Taken together with previous investigations, these observations are consistent with a model of decreased TCA activity with concomitant increase in lipogenesis induced by SR. These tissue-specific mechanistic insights into metabolic effects of SR provide a springboard to future metabolic intervention studies.

ACSS2-mediated acetyl-CoA synthesis from acetate is necessary for human cytomegalovirus infection

Significance Viruses rely completely on host cell metabolism to provide the building blocks and energy required for producing progeny virions. Infection by human cytomegalovirus (HCMV) induces significant alterations in glucose metabolism by increasing glucose uptake and glycolysis as well as redirecting glucose carbon to support the synthesis of biomolecules such as lipids. The significance of acetate as a nutrient has been ignored for a long period. Our studies show that glucose carbon can be converted to acetate and used to make cytosolic acetyl-CoA by acetyl-CoA synthetase short-chain family member 2 (ACSS2) for lipid synthesis, which is important for HCMV-induced lipogenesis and the viral growth. The study provides greater understanding of HCMV pathogenesis and suggests strategies to develop antiviral therapies. Recent studies have shown that human cytomegalovirus (HCMV) can induce a robust increase in lipid synthesis which is critical for the success of infection. In mammalian cells the central precursor for lipid biosynthesis, cytosolic acetyl CoA (Ac-CoA), is produced by ATP-citrate lyase (ACLY) from mitochondria-derived citrate or by acetyl-CoA synthetase short-chain family member 2 (ACSS2) from acetate. It has been reported that ACLY is the primary enzyme involved in making cytosolic Ac-CoA in cells with abundant nutrients. However, using CRISPR/Cas9 technology, we have shown that ACLY is not essential for HCMV growth and virally induced lipogenesis. Instead, we found that in HCMV-infected cells glucose carbon can be used for lipid synthesis by both ACLY and ACSS2 reactions. Further, the ACSS2 reaction can compensate for the loss of ACLY. However, in ACSS2-KO human fibroblasts both HCMV-induced lipogenesis from glucose and viral growth were sharply reduced. This reduction suggests that glucose-derived acetate is being used to synthesize cytosolic Ac-CoA by ACSS2. Previous studies have not established a mechanism for the production of acetate directly from glucose metabolism. Here we show that HCMV-infected cells produce more glucose-derived pyruvate, which can be converted to acetate through a nonenzymatic mechanism.

Time is ripe: maturation of metabolomics in chronobiology.

Sleep and circadian rhythms studies have recently benefited from metabolomics analyses, uncovering new connections between chronobiology and metabolism. From untargeted mass spectrometry to quantitative nuclear magnetic resonance spectroscopy, a diversity of analytical approaches has been applied for biomarker discovery in the field. In this review we consider advances in the application of metabolomics technologies which have uncovered significant effects of sleep and circadian cycles on several metabolites, namely phosphatidylcholine species, medium-chain carnitines, and aromatic amino acids. Study design and data processing measures essential for detecting rhythmicity in metabolomics data are also discussed. Future developments in these technologies are anticipated vis-à-vis validating early findings, given metabolomics has only recently entered the ring with other systems biology assessments in chronometabolism studies.

Altered diurnal states in insomnia reflect peripheral hyperarousal and metabolic desynchrony: a preliminary study

Study Objectives Insomnia is a common sleep disorder that is associated with a range of adverse outcomes. Patients with insomnia exhibit hyperarousal in multiple domains, including an elevated metabolic rate, but specific metabolic molecular perturbations are unknown. Furthermore, objective clinical markers of insomnia are not available and current assessment of pathological extent relies on self-report. Here, we provide preliminary evidence that chronic insomnia is remarkably reflected in the periphery through detailed metabolic assessments. Methods Serum from confirmed patients with insomnia and matched good sleepers (n = 15 per group) was sampled at high temporal resolution (every 2 hr over 48 hr). Food intake was controlled by providing hourly isocaloric snacks, and sleep architecture was assessed by overnight polysomnography. Quantitative metabolic assessments were conducted using nuclear magnetic resonance spectroscopy. Results Global metabolic profiles differentiated patients with insomnia from healthy controls, with elevated amino acid and energy metabolites and reduced branched-chain amino acid catabolic products. Strikingly, branched-chain amino acid catabolism was found to be specifically altered during the night with ~10 per cent increased accumulation of glucose in insomnia patients. Rhythmicity analysis revealed 11 metabolites that cycled diurnally across both groups, with phase advances noted for acetone and delays for lactate and branched-chain amino acids and their products. Conclusions These preliminary observations suggest that insomnia is associated with quantitative metabolic dysregulation and supports the hyperarousal hypothesis. Furthermore, we posit that these changes lead to a state of metabolic desynchrony in insomnia that is involved in the pathophysiology of the disorder and/or mediates its impact on health outcomes. Clinical Trials Registration NCT01957111.

SOFAST-HMQC—an efficient tool for metabolomics

AbstractNuclear magnetic resonance (NMR)-based metabolomics relies mostly on 1D NMR; however, the technique is limited by overlap of the signals from the metabolites. In order to circumvent this problem, 2D 1H-13C correlation spectroscopy techniques are often used. However owing to poorer natural abundance and gyromagnetic ratio of 13C, the acquisition time for 2D 1H-13C heteronuclear single quantum coherence spectroscopy (HSQC) is long. This makes it almost impossible to be used in high throughput study. We have reported the application of selective optimized flip angle short transient (SOFAST) technique coupled to heteronuclear multiple quantum correlation (HMQC) along with nonlinear sampling (NUS) in urine and serum samples. This technique takes sevenfold less experimental time than the conventional 1H-13C HSQC experiment with retention of almost all molecular information. Hence, this can be used for high throughput study. Graphical abstractSOFAST-HMQC is a two-dimensional NMR technique that significantly decreases experimental time without loss of information. This technique is applied in complex biofluid samples that are used for high throughput metabolomics studies and shows promise of better information recovery than conventional two-dimensional NMR technique in shorter time.

CHAPTER 3:NMR Spectroscopy of Urine

NMR spectroscopy of urine is a fertile bioanalytical approach for a wide range of studies in areas such as toxicity, drug development, molecular epidemiology, disease diagnosis, and nutrition. In this chapter, technical concerns critical to the design and execution of urinary NMR experiments are explored. Beginning with the chemical characteristics of urinary NMR spectra, we discuss the history of urinary NMR metabolomics through studies of toxicity and its suitability as a platform for large-scale studies due to high reproducibility and robustness. With respect to experimental design, a detailed discussion of validated urine collection procedures for both human and other animal model experimental systems is provided along with procedures for the use of preservatives and storage. We explore specific issues in the acquisition of urinary NMR experiments, such as the choice of pulse program and solvent suppression. Data pre-processing techniques, such as spectral binning, quantitative peak-fitting, and full-spectrum approaches, as input to subsequent chemometric evaluation of NMR spectra are detailed. Moving towards applications, we review illustrative biological examples of NMR spectroscopy of urine to studies of normal variation and non-healthy phenotypes. Finally, we discuss emerging challenges in biomarker discovery as well as the emerging field of pharmacometabonomics.