Alterations in tricarboxylic acid (TCA) cycle metabolism are associated with hepatic metabolic disorders. Elevated hepatic acetate concentrations, often attributed to high caloric intake, are recognized as a pivotal factor in the etiology of obesity and metabolic syndrome. Therefore, the assessment of acetate breakdown and TCA cycle activity plays a central role in understanding the impact of diet-induced alterations on liver metabolism. Magnetic resonance-based deuterium metabolic imaging (DMI) could help to unravel the underlying mechanisms involved in disease development and progression, however, the application of conventional deuterated glucose does not lead to substantial enrichment in hepatic glutamine and glutamate. This study aimed to demonstrate the feasibility of DMI for tracking deuterated acetate breakdown via the TCA cycle in lean and diet-induced fatty liver (FL) rats using 3D DMI after an intraperitoneal infusion of sodium acetate-d3 at 9.4T. Localized and nonlocalized liver spectra acquired at 10 time points post-injection over a 130-min study revealed similar intrahepatic acetate uptake in both animal groups (AUCFL = 717.9 ± 131.1 mM▯min-1, AUClean = 605.1 ± 119.9 mM▯min-1, p = 0.62). Metabolic breakdown could be observed in both groups with an emerging glutamine/glutamate (Glx) peak as a downstream metabolic product (AUCFL = 113.6 ± 23.8 mM▯min-1, AUClean = 136.7 ± 41.7 mM▯min-1, p = 0.68). This study showed the viability of DMI for tracking substrate flux through the TCA cycle, underscoring its methodological potential for imaging metabolic processes in the body.

• MeSH
• Acetates metabolism   MeSH
• Metabolic Flux Analysis MeSH
• Citric Acid Cycle * MeSH
• Deuterium * MeSH
• Liver * metabolism   diagnostic imaging   MeSH
• Rats MeSH
• Magnetic Resonance Imaging MeSH
• Rats, Sprague-Dawley MeSH
• Rats, Wistar MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

OBJECTIVE: Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. METHODS: We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells. RESULTS: Glucose stimulation significantly increases ROS levels in β-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. CONCLUSIONS: Proper functioning of pancreatic β-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic β-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in β-cell function.

• MeSH
• Insulin-Secreting Cells * metabolism   drug effects   MeSH
• Adaptation, Physiological physiology   MeSH
• Glucose * metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Oxidation-Reduction * MeSH
• Protein Processing, Post-Translational MeSH
• Reactive Oxygen Species * metabolism   MeSH
• Insulin Secretion drug effects   physiology   MeSH
• Signal Transduction * physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Tumor cells often adapt to amino acid deprivation through metabolic rewiring, compensating for the loss with alternative amino acids/substrates. We have described such a scenario in leukemic cells treated with L-asparaginase (ASNase). Clinical effect of ASNase is based on nutrient stress achieved by its dual enzymatic action which leads to depletion of asparagine and glutamine and is accompanied with elevated aspartate and glutamate concentrations in serum of acute lymphoblastic leukemia patients. We showed that in these limited conditions glutamate uptake compensates for the loss of glutamine availability. Extracellular glutamate flux detection confirms its integration into the TCA cycle and its participation in nucleotide and glutathione synthesis. Importantly, it is glutamate-driven de novo synthesis of glutathione which is the essential metabolic pathway necessary for glutamate's pro-survival effect. In vivo findings support this effect by showing that inhibition of glutamate transporters enhances the therapeutic effect of ASNase. In summary, ASNase induces elevated extracellular glutamate levels under nutrient stress, which leads to a rewiring of intracellular glutamate metabolism and has a negative impact on ASNase treatment.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma drug therapy   metabolism   pathology   MeSH
• Asparaginase * pharmacology   metabolism   MeSH
• Citric Acid Cycle drug effects   MeSH
• Glutamine metabolism   MeSH
• Glutathione * metabolism   MeSH
• Glutamic Acid * metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Antineoplastic Agents pharmacology   MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The recognition that rapidly proliferating cancer cells rely heavily on glutamine for their survival and growth has renewed interest in the development of glutamine antagonists for cancer therapy. Glutamine plays a pivotal role as a carbon source for synthesizing lipids and metabolites through the TCA cycle, as well as a nitrogen source for synthesis of amino acid and nucleotides. Numerous studies have explored the significance of glutamine metabolism in cancer, providing a robust rationale for targeting this metabolic pathway in cancer treatment. The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) has been explored as an anticancer therapeutic for nearly six decades. Initial investigations revealed remarkable efficacy in preclinical studies and promising outcomes in early clinical trials. However, further advancement of DON was hindered due to dose-limiting gastrointestinal (GI) toxicities as the GI system is highly dependent on glutamine for regulating growth and repair. In an effort to repurpose DON and mitigate gastrointestinal (GI) toxicity concerns, prodrug strategies were utilized. These strategies aimed to enhance the delivery of DON to specific target tissues, such as tumors and the central nervous system (CNS), while sparing DON delivery to normal tissues, particularly the GI tract. When administered at low daily doses, optimized for metabolic inhibition, these prodrugs exhibit remarkable effectiveness without inducing significant toxicity to normal tissues. This approach holds promise for overcoming past challenges associated with DON, offering an avenue for its successful utilization in cancer treatment.

• MeSH
• Diazooxonorleucine * pharmacology   therapeutic use   MeSH
• Glutamine metabolism   MeSH
• Humans MeSH
• Neoplasms * drug therapy   metabolism   MeSH
• Prodrugs * pharmacology   therapeutic use   MeSH
• Antineoplastic Agents pharmacology   therapeutic use   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist that suppresses cancer cell metabolism but concurrently enhances the metabolic fitness of tumor CD8+ T cells. DON showed promising efficacy in clinical trials; however, its development was halted by dose-limiting gastrointestinal (GI) toxicities. Given its clinical potential, we designed DON peptide prodrugs and found DRP-104 [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] that was preferentially bioactivated to DON in tumor while bioinactivated to an inert metabolite in GI tissues. In drug distribution studies, DRP-104 delivered a prodigious 11-fold greater exposure of DON to tumor versus GI tissues. DRP-104 affected multiple metabolic pathways in tumor, including decreased glutamine flux into the TCA cycle. In efficacy studies, both DRP-104 and DON caused complete tumor regression; however, DRP-104 had a markedly improved tolerability profile. DRP-104's effect was CD8+ T cell dependent and resulted in robust immunologic memory. DRP-104 represents a first-in-class prodrug with differential metabolism in target versus toxicity tissue. DRP-104 is now in clinical trials under the FDA Fast Track designation.

• MeSH
• CD8-Positive T-Lymphocytes metabolism   MeSH
• Diazooxonorleucine pharmacology   therapeutic use   MeSH
• Glutamine metabolism   MeSH
• Enzyme Inhibitors therapeutic use   MeSH
• Humans MeSH
• Neoplasms * drug therapy   MeSH
• Prodrugs * pharmacology   therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Cisplatin (cis-diamminedichloroplatinum II; CDDP) is a widely used cytostatic agent; however, it tends to promote kidney and liver disease, which are a major signs of drug-induced toxicity. Platinum compounds are often presented as alternative therapeutics and subsequently easily dispersed in the environment as contaminants. Due to the major roles of the liver and kidneys in removing toxic materials from the human body, we performed a comparative study of the amino acid profiles in chicken liver and kidneys before and after the application of CDDP and platinum nanoparticles (PtNPs-10 and PtNPs-40). The treatment of the liver with the selected drugs affected different amino acids; however, Leu and Arg were decreased after all treatments. The treatment of the kidneys with CDDP mostly affected Val; PtNPs-10 decreased Val, Ile and Thr; and PtNPs-40 affected only Pro. In addition, we tested the same drugs on two healthy cell lines, HaCaT and HEK-293, and ultimately explored the amino acid profiles in relation to the tricarboxylic acid cycle (TCA) and methionine cycle, which revealed that in both cell lines, there was a general increase in amino acid concentrations associated with changes in the concentrations of the metabolites of these cycles.

• Publication type
• Journal Article MeSH

The Entner-Doudoroff pathway (ED-P) was established in 2016 as the fourth glycolytic pathway in Synechocystis sp. PCC 6803. ED-P consists of two reactions, the first catalyzed by 6-phosphogluconate dehydratase (EDD), the second by keto3-deoxygluconate-6-phosphate aldolase/4-hydroxy-2-oxoglutarate aldolase (EDA). ED-P was previously concluded to be a widespread (∼92%) pathway among cyanobacteria, but current bioinformatic analysis estimated the occurrence of ED-P to be either scarce (∼1%) or uncommon (∼47%), depending if dihydroxy-acid dehydratase (ilvD) also functions as EDD (currently assumed). Thus, the biochemical characterization of ilvD is a task pending to resolve this uncertainty. Next, we have provided new insights into several single and double glycolytic mutants based on kinetic model of central carbon metabolism of Synechocystis. The model predicted that silencing 6-phosphogluconate dehydrogenase (gnd) could be coupled with ∼90% down-regulation of G6P-dehydrogenase, also limiting the metabolic flux via ED-P. Furthermore, our metabolic flux estimation implied that growth impairment linked to silenced EDA under mixotrophic conditions is not caused by diminished carbon flux via ED-P but rather by a missing mechanism related to the role of EDA in metabolism. We proposed two possible, mutually non-exclusive explanations: (i) Δeda leads to disrupted carbon catabolite repression, regulated by 2-keto3-deoxygluconate-6-phosphate (ED-P intermediate), and (ii) EDA catalyzes the interconversion between glyoxylate and 4-hydroxy-2-oxoglutarate + pyruvate in the proximity of TCA cycle, possibly effecting the levels of 2-oxoglutarate under Δeda. We have also proposed a new pathway from EDA toward proline, which could explain the proline accumulation under Δeda. In addition, the presented in silico method provides an alternative to 13C metabolic flux analysis for marginal metabolic pathways around/below the threshold of ultrasensitive LC-MS. Finally, our in silico analysis provided alternative explanations for the role of ED-P in Synechocystis while identifying some severe uncertainties.

• Publication type
• Journal Article MeSH

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

• MeSH
• Adenylate Kinase * metabolism   MeSH
• Cyclin-Dependent Kinase 5 metabolism   MeSH
• Energy Metabolism MeSH
• Phosphorylation MeSH
• Glycogen Synthase Kinase 3 metabolism   MeSH
• Mice MeSH
• Carcinoma, Neuroendocrine * MeSH
• Succinates MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

BACKGROUND: Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. RESULTS: We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5'-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. CONCLUSIONS: This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

• MeSH
• Amoeba * genetics   metabolism   MeSH
• Anaerobiosis MeSH
• Archamoebae * genetics   metabolism   MeSH
• Genomics MeSH
• Humans MeSH
• Mitochondria metabolism   MeSH
• Peroxisomes metabolism   MeSH
• Saccharomyces cerevisiae MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

• MeSH
• Citric Acid Cycle drug effects   MeSH
• Glucose metabolism   MeSH
• Insect Vectors parasitology   MeSH
• Tsetse Flies drug effects   parasitology   MeSH
• Oxidation-Reduction drug effects   MeSH
• Proline metabolism   pharmacology   MeSH
• RNA Interference physiology   MeSH
• Trypanosoma brucei brucei drug effects   metabolism   MeSH
• Trypanosoma drug effects   metabolism   MeSH
• Trypanosomiasis, African drug therapy   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH