ROS and hypoxia signaling regulate periodic metabolic arousal during insect dormancy to coordinate glucose, amino acid, and lipid metabolism.

Metabolic suppression is a hallmark of animal dormancy that promotes overall energy savings. Some diapausing insects and some mammalian hibernators have regular cyclic patterns of substantial metabolic depression alternating with periodic arousal where metabolic rates increase dramatically. Previous studies, largely in mammalian hibernators, have shown that periodic arousal is driven by an increase in aerobic mitochondrial metabolism and that many molecules related to energy metabolism fluctuate predictably across periodic arousal cycles. However, it is still not clear how these rapid metabolic shifts are regulated. We first found that diapausing flesh fly pupae primarily use anaerobic glycolysis during metabolic depression but engage in aerobic respiration through the tricarboxylic acid cycle during periodic arousal. Diapausing pupae also clear anaerobic by-products and regenerate many metabolic intermediates depleted in metabolic depression during arousal, consistent with patterns in mammalian hibernators. We found that decreased levels of reactive oxygen species (ROS) induced metabolic arousal and elevated ROS extended the duration of metabolic depression. Our data suggest ROS regulates the timing of metabolic arousal by changing the activity of two critical metabolic enzymes, pyruvate dehydrogenase and carnitine palmitoyltransferase I by modulating the levels of hypoxia inducible transcription factor (HIF) and phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Our study shows that ROS signaling regulates periodic arousal in our insect diapasue system, suggesting the possible importance ROS for regulating other types of of metabolic cycles in dormancy as well.

DFT investigation of coupling constant anomalies in substituted ß-lactams.

ß-lactams are a chemically diverse group of molecules with a wide range of biological activities. Having recently observed curious trends in 2JHH coupling values in studies on this structural class, we sought to obtain a more comprehensive understanding of these diagnostic NMR parameters, specifically interrogating 1JCH, 2JCH, and 2JHH, to differentiate 3- and 4-monosubstituted ß-lactams. Further investigation using computational chemistry methods was employed to explore the geometric and electronic origins for the observed and calculated differences between the two substitution patterns.

Segmented Flow Strategies for Integrating Liquid Chromatography-Mass Spectrometry with Nuclear Magnetic Resonance for Lipidomics.

Building an accurate lipid inventory relies on coordinated information from orthogonal analytical capabilities. Integrating the familiar workflow of liquid chromatography (LC), high-resolution mass spectrometry (HRMS), and tandem mass spectrometry (MS/MS) with proton nuclear magnetic resonance spectroscopy (1H NMR) would be ideal for building that inventory. For absolute lipid structural elucidation, LC-HRMS/MS can provide lower-level structural information with superior sensitivity, while 1H NMR can provide invaluable higher-order structural information for the disambiguation of isomers with absolute chemical specificity. Digitization of the LC eluent followed by splitting the microfractions into two flow paths in a defined ratio for HRMS/MS and NMR would be the ideal strategy to permit correlation of the MS and NMR data as a function of chromatographic retention time. Here, we report an active segmentation platform to transform analytical flow rate LC eluent into parallel microliter segmented flow queues for high confidence correlation of the MS, MS/MS, and NMR data. The practical details in implementing this strategy to achieve an integrated LC-MS-NMR platform are presented, including the development of an active segmentation technology using a four-port two-way valve to transform the LC eluent into parallel segmented flows for online MS analysis followed by offline segment-specific 1H NMR and optimization of the detector response toward segmented flow. To demonstrate the practicality of this novel platform, it was tested using lipid mixture samples.

Enrichment of hepatic glycogen and plasma glucose from H218 O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice.

Deuterated water (2 H2 O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of 2 H can distort metabolic information and mediate toxicity. 18 O-water (H2 18 O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well-defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H2 18 O during overnight feeding and 18 O-enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by 13 C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG 18 O-enrichments were quantified by MS. Blood glucose 18 O-enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG 18 O-enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H2 18 O informs hepatic carbohydrate biosynthesis in similar detail to 2 H2 O but without KIE-associated risks.

Deuterated water imaging of the rat brain following metabolism of [2 H7 ]glucose.

PURPOSE: To determine whether deuterated water (HDO) generated from the metabolism of [2 H7 ]glucose is a sensitive biomarker of cerebral glycolysis and oxidative flux. METHODS: A bolus of [2 H7 ]glucose was injected through the tail vein at 1.95 g/kg into Sprague-Dawley rats. A 2 H surface coil was placed on top of the head to record 2 H spectra of the brain every 1.3 minutes to measure glucose uptake and metabolism to HDO, lactate, and glutamate/glutamine. A two-point Dixon method based on a gradient-echo sequence was used to reconstruct deuterated glucose and water (HDO) images selectively. RESULTS: The background HDO signal could be detected and imaged before glucose injection. The 2 H NMR spectra showed arrival of [2 H7 ]glucose and its metabolism in a time-dependent manner. A ratio of the HDO to glutamate/glutamine resonances demonstrates a pseudo-steady state following injection, in which cerebral metabolism dominates wash-in of HDO generated by peripheral metabolism. Brain spectroscopy reveals that HDO generation is linear with lactate and glutamate/glutamine appearance in the appropriate pseudo-steady state window. Selective imaging of HDO and glucose is easily accomplished using a gradient-echo method. CONCLUSION: Metabolic imaging of HDO, as a marker of glucose, lactate, and glutamate/glutamine metabolism, has been shown here for the first time. Cerebral glucose metabolism can be assessed efficiently using a standard gradient-echo sequence that provides superior in-plane resolution compared with CSI-based techniques.

15 N-carnitine, a novel endogenous hyperpolarized MRI probe with long signal lifetime.

PURPOSE: The purpose of this study was to investigate hyperpolarization and in vivo imaging of [15 N]carnitine, a novel endogenous MRI probe with long signal lifetime. METHODS: L-[15 N]carnitine-d9 was hyperpolarized by the method of dynamic nuclear polarization followed by rapid dissolution. The T1 signal lifetimes were estimated in aqueous solution and in vivo following intravenous injection in rats, using a custom-built dual-tuned 15 N/1 H RF coil at 4.7 T. 15 N chemical shift imaging and 15 N fast spin-echo images of rat abdomen were acquired 3 minutes after [15 N]carnitine injection. RESULTS: Estimated T1 times of [15 N]carnitine at 4.7 T were 210 seconds (in H2 O) and 160 seconds (in vivo), with an estimated polarization level of 10%. Remarkably, the [15 N]carnitine coherence was detectable in rat abdomen for 5 minutes after injection for the nonlocalized acquisition. No downstream metabolites were detected on localized or nonlocalized 15 N spectra. Diffuse liver enhancement was detected on 15 N fast spin-echo imaging 3 minutes after injection, with mean hepatic SNR of 18 ± 5 at a spatial resolution of 4 × 4 mm. CONCLUSION: This study showed the feasibility of hyperpolarizing and imaging the biodistribution of HP [15 N]carnitine.

Branched chain amino acids and carbohydrate restriction exacerbate ketogenesis and hepatic mitochondrial oxidative dysfunction during NAFLD.

Mitochondrial adaptation during non-alcoholic fatty liver disease (NAFLD) include remodeling of ketogenic flux and sustained tricarboxylic acid (TCA) cycle activity, which are concurrent to onset of oxidative stress. Over 70% of obese humans have NAFLD and ketogenic diets are common weight loss strategies. However, the effectiveness of ketogenic diets toward alleviating NAFLD remains unclear. We hypothesized that chronic ketogenesis will worsen metabolic dysfunction and oxidative stress during NAFLD. Mice (C57BL/6) were kept (for 16-wks) on either a low-fat, high-fat, or high-fat diet supplemented with 1.5X branched chain amino acids (BCAAs) by replacing carbohydrate calories (ketogenic). The ketogenic diet induced hepatic lipid oxidation and ketogenesis, and produced multifaceted changes in flux through the individual steps of the TCA cycle. Higher rates of hepatic oxidative fluxes fueled by the ketogenic diet paralleled lower rates of de novo lipogenesis. Interestingly, this metabolic remodeling did not improve insulin resistance, but induced fibrogenic genes and inflammation in the liver. Under a chronic "ketogenic environment," the hepatocyte diverted more acetyl-CoA away from lipogenesis toward ketogenesis and TCA cycle, a milieu which can hasten oxidative stress and inflammation. In summary, chronic exposure to ketogenic environment during obesity and NAFLD has the potential to aggravate hepatic mitochondrial dysfunction.

Quantitative Analysis of Bioactive Carbazole Alkaloids in Murraya koenigii (L.) from Six Different Climatic Zones of India Using UPLC/MS/MS and Their Principal Component Analysis.

Murraya koenigii (L.) Spreng (Curry leaf) is a commercially important medicinal plant in South Asia, containing therapeutically valuable carbazole alkaloids (CAs). Thus, the quantitative evaluation of these compounds from different climatic zones of India are an important aspect for quality assessment and economic isolation of targeted compounds from the plant. In this study, quantitative estimation of CAs among 34 Indian natural populations of M. koenigii was assessed using UPLC/MS/MS. The collected populations represent the humid subtropical, tropical wet & dry, tropical wet, semi-arid, arid, and montane climatic zones of India. A total of 11 CAs viz. koenine-I, murrayamine A, koenigine, koenimbidine, koenimbine, O-methylmurrayamine A, girinimbine, mahanine, 8,8''-biskoenigine, isomahanimbine, and mahanimbine were quantified using multiple reaction monitoring (MRM) experiments within 5.0 min. The respective range for natural abundance of CAs were observed as 0.097-1.222, 0.092-5.014, 0.034-0.661, 0.010-1.673, 0.013-7.336, 0.010-0.310, 0.010-0.114, 0.049-5.288, 0.031-1.731, 0.491-3.791, and 0.492-5.399 mg/g in leaves of M. koenigii. The developed method shown linearity regression coefficient (r2 >0.9995), LOD (0.003-0.248 ng/mL), LOQ (0.009-0.754 ng/mL), and the recovery was between 88.803-103.729 %. The bulk of these CAs were recorded in their highest concentrations in the humid subtropical zone, followed by the tropical wet & dry zones of India. Further, principal component analysis (PCA) was performed which differentiated the climatic zones according to the dominant and significant CAs contents within the populations. The study concludes that the method established is simple, rapid, with high sample throughput, and can be used as a tool for commercial purposes and quality control of M. koenigii.

Synthesis of S-(-)-5,6-Dihydrocanthin-4-ones via a Triple Cooperative Catalysis-Mediated Domino Reaction.

An enantioselective synthesis of S-(-)-5,6-dihydrocanthin-4-ones via a triple cooperative catalysis-mediated domino reaction having a broad substrate scope is reported. The reaction between substituted 1-formyl-9H-ß-carbolines and terminal alkynes in the presence of catalytic amounts of Jorgensen-Hayashi catalyst, copper iodide, and Hunig base proceeded via a multicascade route, affording the title compounds in good yields and excellent ees with interesting mechanistic features. These compounds were assessed for in vitro antiplasmodial activity against P. falciparum strains. Additionally, 5,6-dihydrocanthin-4-ones are demonstrated to be a versatile precursor to different fused ß-carboline derivatives via simple synthetic transformations.

Identification of gallic acid based glycoconjugates as a novel tubulin polymerization inhibitors.

A novel class of gallic acid based glycoconjugates were designed and synthesized as potential anticancer agents. Among all the compounds screened, compound 2a showed potent anticancer activity against breast cancer cells. The latter resulted in tubulin polymerization inhibition and induced G2/M cell cycle arrest, generation of reactive oxygen species, mitochondrial depolarization and subsequent apoptosis in breast cancer cells. In addition, ultraviolet-visible spectroscopy and fluorescence quenching studies of the compound with tubulin confirmed direct interaction of compounds with tubulin. Molecular modeling studies revealed that it binds at the colchicine binding site in tubulin. Further, 2a also exhibited potent in vivo anticancer activity in LA-7 syngeneic rat mammary tumor model. Current data projects its strong candidature to be developed as anticancer agent.

Cyclodextrin in drug delivery: Exploring scaffolds, properties, and cutting-edge applications.

Cyclodextrins (CDs) are unique cyclic compounds that can form inclusion complexes via host-guest complexation with a wide range of molecules, thereby altering their physicochemical properties. These molecules offer the formation of inclusion complexes without the formation of covalent bonds, making them suitable for a variety of applications in pharmaceutical and biomedical fields. Due to their supramolecular host-guest properties, CDs are being utilized in the fabrication of biomaterials, metal-organic frameworks, and nano-drug carriers. Additionally, CDs in combination with biomolecules are biocompatible and can deliver nano to macromolecules at the site of drug actions. However, the availability of free hydroxyl groups and a simple crosslinking process for supramolecular fabrication show immense opportunities for researchers in the field of tissue engineering and biomedical applications. In this review article, we have covered the historical development, various types of chemical frameworks, unique chemical and physical properties, and important applications of CDs in drug delivery and biomedical sciences.

Exploring the Significance, Extraction, and Characterization of Plant-Derived Secondary Metabolites in Complex Mixtures.

Secondary metabolites are essential components for the survival of plants. Secondary metabolites in complex mixtures from plants have been adopted and documented by different traditional medicinal systems worldwide for the treatment of various human diseases. The extraction strategies are the key components for therapeutic development from natural sources. Polarity-dependent solvent-selective extraction, acidic and basic solution-based extraction, and microwave- and ultrasound-assisted extraction are some of the most important strategies for the extraction of natural products from plants. The method needs to be optimized to isolate a specific class of compounds. Therefore, to establish the mechanism of action, the characterization of the secondary metabolites, in a mixture or in their pure forms, is equally important. LC-MS, GC-MS, and extensive NMR spectroscopic strategies are established techniques for the profiling of metabolites in crude extracts. Various protocols for the extraction and characterization of a wide range of classes of compounds have been developed by various research groups and are described in this review. Additionally, the possible means of characterizing the compounds in the mixture and their uniqueness are also discussed. Hyphenated techniques are crucial for profiling because of their ability to analyze a vast range of compounds. In contrast, inherent chemical shifts make NMR an indispensable tool for structure elucidation in complex mixtures.

Age-Related Differences in Affective Behaviors in Mice: Possible Role of Prefrontal Cortical-Hippocampal Functional Connectivity and Metabolomic Profiles.

The differential expression of emotional reactivity from early to late adulthood may involve maturation of prefrontal cortical responses to negative valence stimuli. In mice, age-related changes in affective behaviors have been reported, but the functional neural circuitry warrants further investigation. We assessed age variations in affective behaviors and functional connectivity in male and female C57BL6/J mice. Mice aged 10, 30 and 60 weeks (wo) were tested over 8 weeks for open field activity, sucrose preference, social interactions, fear conditioning, and functional neuroimaging. Prefrontal cortical and hippocampal tissues were excised for metabolomics. Our results indicate that young and old mice differ significantly in affective behavioral, functional connectome and prefrontal cortical-hippocampal metabolome. Young mice show a greater responsivity to novel environmental and social stimuli compared to older mice. Conversely, late middle-aged mice (60wo group) display variable patterns of fear conditioning and with re-testing with a modified context. Functional connectivity between a temporal cortical/auditory cortex network and subregions of the anterior cingulate cortex and ventral hippocampus, and a greater network modularity and assortative mixing of nodes was stronger in young versus older adult mice. Metabolome analyses identified differences in several essential amino acids between 10wo mice and the other age groups. The results support differential expression of 'emotionality' across distinct stages of the mouse lifespan involving greater prefrontal-hippocampal connectivity and neurochemistry.

Inactivation of mitochondrial MUL1 E3 ubiquitin ligase deregulates mitophagy and prevents diet-induced obesity in mice.

Obesity is a growing epidemic affecting millions of people worldwide and a major risk factor for a multitude of chronic diseases and premature mortality. Accumulating evidence suggests that mitochondria have a profound role in diet-induced obesity and the associated metabolic changes, but the molecular mechanisms linking mitochondria to obesity remain poorly understood. Our studies have identified a new function for mitochondrial MUL1 E3 ubiquitin ligase, a protein known to regulate mitochondrial dynamics and mitophagy, in the control of energy metabolism and lipogenesis. Genetic deletion of Mul1 in mice impedes mitophagy and presents a metabolic phenotype that is resistant to high-fat diet (HFD)-induced obesity and metabolic syndrome. Several metabolic and lipidomic pathways are perturbed in the liver and white adipose tissue (WAT) of Mul1(-/-) animals on HFD, including the one driven by Stearoyl-CoA Desaturase 1 (SCD1), a pivotal regulator of lipid metabolism and obesity. In addition, key enzymes crucial for lipogenesis and fatty acid oxidation such as ACC1, FASN, AMPK, and CPT1 are also modulated in the absence of MUL1. The concerted action of these enzymes, in the absence of MUL1, results in diminished fat storage and heightened fatty acid oxidation. Our findings underscore the significance of MUL1-mediated mitophagy in regulating lipogenesis and adiposity, particularly in the context of HFD. Consequently, our data advocate the potential of MUL1 as a therapeutic target for drug development in the treatment of obesity, insulin resistance, NAFLD, and cardiometabolic diseases.

Age-related differences in affective behaviors in mice: possible role of prefrontal cortical-hippocampal functional connectivity and metabolomic profiles.

Introduction: The differential expression of emotional reactivity from early to late adulthood may involve maturation of prefrontal cortical responses to negative valence stimuli. In mice, age-related changes in affective behaviors have been reported, but the functional neural circuitry warrants further investigation. Methods: We assessed age variations in affective behaviors and functional connectivity in male and female C57BL6/J mice. Mice aged 10, 30 and 60 weeks (wo) were tested over 8 weeks for open field activity, sucrose preference, social interactions, fear conditioning, and functional neuroimaging. Prefrontal cortical and hippocampal tissues were excised for metabolomics. Results: Our results indicate that young and old mice differ significantly in affective behavioral, functional connectome and prefrontal cortical-hippocampal metabolome. Young mice show a greater responsivity to novel environmental and social stimuli compared to older mice. Conversely, late middle-aged mice (60wo group) display variable patterns of fear conditioning and during re-testing in a modified context. Functional connectivity between a temporal cortical/auditory cortex network and subregions of the anterior cingulate cortex and ventral hippocampus, and a greater network modularity and assortative mixing of nodes was stronger in young versus older adult mice. Metabolome analyses identified differences in several essential amino acids between 10wo mice and the other age groups. Discussion: The results support differential expression of 'emotionality' across distinct stages of the mouse lifespan involving greater prefrontal-hippocampal connectivity and neurochemistry.

Detecting altered hepatic lipid oxidation by MRI in an animal model of MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing annually and affects over a third of US adults. MASLD can progress to metabolic dysfunction-associated steatohepatitis (MASH), characterized by severe hepatocyte injury, inflammation, and eventual advanced fibrosis or cirrhosis. MASH is predicted to become the primary cause of liver transplant by 2030. Although the etiology of MASLD/MASH is incompletely understood, dysregulated fatty acid oxidation is implicated in disease pathogenesis. Here, we develop a method for estimating hepatic ß-oxidation from the metabolism of [D15]octanoate to deuterated water and detection with deuterium magnetic resonance methods. Perfused livers from a mouse model of MASLD reveal dysregulated hepatic ß-oxidation, findings that corroborate in vivo imaging. The high-fat-diet-induced MASLD mouse studies indicate that decreased ß-oxidative efficiency in the fatty liver could serve as an indicator of MASLD progression. Furthermore, our method provides a clinically translatable imaging approach for determining hepatic ß-oxidation efficiency.

Design and Discovery of Water-Soluble Benzooxaphosphole-Based Ligands for Hindered Suzuki-Miyaura Coupling Reactions with Low Catalyst Load.

We report a new class of highly effective, benzooxaphosphole-based, water-soluble ligands in the application of Suzuki-Miyaura cross-coupling reactions for sterically hindered substrates in aqueous media. The catalytic activities of the coupling reactions were greatly enhanced by the addition of catalytic amounts of organic phase transfer reagents, such as tetraglyme and tetrabutylammonium bromide. The optimized general protocol can be conducted with a low catalyst load, thereby providing a practical solution for these reactions. The viability of this new Suzuki-Miyaura protocol was demonstrated with various substrates to generate important building blocks, including heterocycles, for the synthesis of biologically active compounds.

Synthesis of triazolo isoquinolines and isochromenes from 2-alkynylbenzaldehyde via domino reactions under transition-metal-free conditions.

We describe two simple straightforward syntheses of triazolo isoquinolines (3) and isochromenes (7) from 2-alkynylbenzaldehydes (1) as a common synthon. The synthetic strategy for 3 involves formation of the (E)-1-(2-nitrovinyl)-2-(alkynyl)benzene species 2 via condensation of synthon 1 with nitromethane followed by a [3 + 2] cycloaddition/extrusion of the nitro group/regioselective 6-endo cyclization domino sequence. In yet another strategy, the synthon 1 was condensed with nitromethane followed by electrophilic iodo cyclization of the resulting 2-nitro-1-(2-(alkynyl)phenyl)ethanol (6) to furnish iodo isochromene derivatives. The salient feature of the above two strategies involves formation of the corresponding heterocycles under metal-free conditions in good yields.

A strategy for the synthesis of anthraquinone-based aryl-C-glycosides.

An efficient and simple strategy for the synthesis of a diverse range of anthraquinone-based aryl-C-glycosides has been developed. It involves the sequential Diels-Alder reaction and oxidative aromatization with the preformed glycosyl diene and dienophiles. The glycosyl dienes were obtained from simple sugars by tandem one-pot substitution and elimination reaction.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.