Tricarboxylic Cycle Research Articles

Fumarate Reduced Mean Arterial Blood Pressure and Improved Renal Outcomes in DOCA/salt Hypertension

The mitochondria, through the tricarboxylic (TCA) acid cycle, converts organic molecules into energy that drives intricate renal blood‐pressure regulatory functions. The TCA cycle intermediaries regulate the metabolic state of the mitochondria and by extension, the redox status of the cell. Recently, fumarase, a major TCA cycle enzyme that converts fumarate to malate, has been linked to L‐arginine metabolism and implicated in the pathophysiology of Dahl‐salt sensitive hypertension. This study aimed to investigate the role of fumarase and L‐arginine in deoxycorticosterone (DOCA)/salt hypertension, a non‐genetic model of hypertension. Hypertension was induced in uninephrectomised rats with DOCA (25 mg/kg) + 1 % NaCL. Mean arterial blood pressure was measured in conscious rats via carotid cannulation. Biochemical and western blot analyses were carried out on homogenized kidney fractions. Fumarate reduced arterial blood pressure (198±5 vs 167±7 mmHg, P<0.01). There were no changes in urinary output and sodium excretion in fumarate‐treated DOCA rats (P>0.05). Superoxide dismutase and catalase activities were higher in the cortex, but not the medulla of DOCA‐hypertensive rats and were exacerbated by fumarate (P<0.01). There was a selective increase in catalase activity in the medulla of fumarate‐treated DOCA‐hypertensive rats (P<0.01). Fumarate also reduced urinary protein excretion in DOCA‐hypertensive rats (23.6 %, P<0.05). Fumarate evoked a selective increase in nitric oxide in the medulla (34.1±2.9 vs 47.3±2.8 nM/μg, P<0.05) of DOCA‐hypertensive rats. Consistent with this, Fumarate also tended to increase arginase activity in the cortex but not the medulla of DOCA‐hypertensive rats. However, there was an increase in expression TGF‐β, an index of fibrosis, in DOCA‐hypertensive rats, which was paradoxically increased in fumarate‐treated rats (P>0.05). In conclusion, fumarate attenuated the hypertension and renal injury but not the fibrosis in DOCA/salt hypertension by mechanisms involving selective reduction of L‐arginine metabolism in the medulla and amelioration of renal oxidative stress.Support or Funding InformationNational Institute of Health grant HL03674 and G12 MD0076056Mean arterial blood changes after 21‐day administration of fumarate to DOCA/salt induced hypertensive rats. Control (n=8), DOCA (n=8), fumarate (n=8). a=P<0.001 when compared to control; b=P<0.01 when compared to DOCA group.Figure 1Nitric oxide produced in homogenized kidneys (cortex and medulla) of DOCA salt induced hypertensive rats after 21‐day fumarate (1 mg/kg) administrationa=P<0.05 when compared to control.Figure 2

Targeting NRF2-Governed Glutathione Synthesis for SDHB-Mutated Pheochromocytoma and Paraganglioma.

Succinate dehydrogenase subunit B (SDHB) deficiency frequently occurs in cluster I pheochromocytomas and paragangliomas (PCPGs). SDHB-mutated PCPGs are characterized by alterations in the electron transport chain, metabolic reprogramming of the tricarboxylic cycle, and elevated levels of reactive oxygen species (ROS). We discovered that SDHB-deficient PCPG cells exhibit increased oxidative stress burden, which leads to elevated demands for glutathione metabolism. Mechanistically, nuclear factor erythroid 2-related factor 2 (NRF2)-guided glutathione de novo synthesis plays a key role in supporting cellular survival and the proliferation of SDHB-knockdown (SDHBKD) cells. NRF2 blockade not only disrupted ROS homeostasis in SDHB-deficient cells but also caused severe cytotoxicity by the accumulation of DNA oxidative damage. Brusatol, a potent NRF2 inhibitor, showed a promising effect in suppressing SDHBKD metastatic lesions in vivo, with prolonged overall survival in mice bearing PCPG allografts. Our findings highlight a novel therapeutic strategy of targeting the NRF2-driven glutathione metabolic pathway against SDHB-mutated PCPG.

Metabolic reprogramming as a feast for virus replication.

Viral replication depends entirely on the energy and biosynthetic precursors supplied by the host cell metabolic network. Viruses actively reprogram host cell metabolism to establish optimal environment for their replication and spread. They stimulate the uptake of extracellular nutrients and predominantly modulate glucose, glutamine, and fatty acid metabolism to support anabolic metabolic pathways. Some viruses activate the process of aerobic glycolysis, divert the glycolytic carbon for biosynthetic reactions, and stimulate glutamine utilization to replenish tricarboxylic cycle intermediates. Others use glutamine carbon to promote de novo fatty acid synthesis, amino acid supply or glutathione production. The unique metabolic signature and different dependence of viral life cycle on the individual metabolic processes is therefore characteristic feature of almost each virus. Deeper understanding of how viruses alter cellular metabolic pathways or their upstream regulatory circuits may lead to development of more effective antiviral treatment strategies based on targeted metabolic inhibition. Keywords: virus infection; metabolism; glycolysis; glutamine metabolism; fatty acid synthesis; metabolic reprogramming; virus-host interaction.

RSM-GA Based Optimization of Bacterial PHA Production and In Silico Modulation of Citrate Synthase for Enhancing PHA Production.

The inexhaustible nature and biodegradability of bioplastics like polyhydroxyalkanoates (PHAs) make them suitable assets to replace synthetic plastics. The eventual fate of these eco-friendly and non-toxic bioplastics relies upon the endeavors towards satisfying cost and, in addition, execution necessity. In this study, we utilized and statistically optimized different food (kitchen-/agro-) waste as a sole carbon/nitrogen source for the production of PHA at a reduced cost, indicating a proficient waste administration procedure. Seven different types of kitchen-/agro-waste were used as unique carbon source and four different types of nitrogen source were used to study their impact on PHA production by Bacillus subtilis MTCC 144. Among four different studied production media, mineral salt medium (MSM) (biomass: 37.7 g/L; cell dry weight: 1.8 g/L; and PHA: 1.54 g/L) was found most suitable for PHA production. Further, carbon and nitrogen components of MSM were optimized using one-factor-at-a-time experiments, and found that watermelon rind (PHA = 12.97 g/L) and pulse peel (PHA = 13.5 g/L) were the most suitable carbon and nitrogen sources, respectively, in terms of PHA (78.60%) recovery. The concentrations of these factors (sources) were statistically optimized using response surface methodology coupled with the genetic algorithm approach. Additionally, in order to enhance microbial PHA production, the interaction of citrate synthase, a key enzyme in the TCA cycle, with different known inhibitors was studied using in silico molecular docking approach. The inhibition of citrate synthase induces the blockage of the tricarboxylic cycle (TCA), thereby increasing the concentration of acetyl-CoA that helps in enhanced PHA production. Molecular docking of citrate synthase with different inhibitors of PubChem database revealed that hesperidin (PubChem compound CID ID 10621), generally present in citrus fruits, is the most efficient inhibitor of the TCA cycle with the binding score of –11.4 and warrants experimental validation. Overall, this study provides an efficient food waste management approach by reducing the production cost and enhancing the production of PHA, thereby lessening our reliance on petroleum-based plastics.

The effect of exercise on intramyocellular acetylcarnitine (AcCtn) concentration in adult growth hormone deficiency (GHD)

To cover increasing energy demands during exercise, tricarboxylic cycle (TCA) flux in skeletal muscle is markedly increased, resulting in the increased formation of intramyocellular acetylcarnitine (AcCtn). We hypothesized that reduced substrate availability within the exercising muscle, reflected by a diminished increase of intramyocellular AcCtn concentration during exercise, might be an underlying mechanism for the impaired exercise performance observed in adult patients with growth hormone deficiency (GHD). We aimed at assessing the effect of 2 hours of moderately intense exercise on intramyocellular AcCtn concentrations, measured by proton magnetic resonance spectroscopy (1H-MRS), in seven adults with GHD compared to seven matched control subjects (CS). Compared to baseline levels AcCtn concentrations significantly increased after 2 hours of exercise, and significantly decreased over the following 24 hours (ANOVA p for effect of time = 0.0023 for all study participants; p = 0.067 for GHD only, p = 0.045 for CS only). AcCtn concentrations at baseline, as well as changes in AcCtn concentrations over time were similar between GHD patients and CS (ANOVA p for group effect = 0.45). There was no interaction between group and time (p = 0.53). Our study suggests that during moderately intense exercise the availability of energy substrate within the exercising muscle is not significantly different in GHD patients compared to CS.

Reductive Stress: New Insights in Physiology and Drug Tolerance of Mycobacterium.

Significance:Mycobacterium tuberculosis (Mtb) encounters reductive stress during its infection cycle. Notably, host-generated protective responses, such as acidic pH inside phagosomes and lysosomes, exposure to glutathione in alveolar hypophase (i.e., a thin liquid lining consisting of surfactant and proteins in the alveolus), and hypoxic environments inside granulomas are associated with the accumulation of reduced cofactors, such as nicotinamide adenine dinucleotide (reduced form), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide (reduced form), and nonprotein thiols (e.g., mycothiol), leading to reductive stress in Mtb cells. Dissipation of this reductive stress is important for survival of the bacterium. If reductive stress is not dissipated, it leads to generation of reactive oxygen species, which may be fatal for the cells. Recent Advances: This review focuses on mechanisms utilized by mycobacteria to sense and respond to reductive stress. Importantly, exposure of Mtb cells to reductive stress leads to growth inhibition, altered metabolism, modulation of virulence, and drug tolerance. Mtb is equipped with thiol buffering systems of mycothiol and ergothioneine to protect itself from various redox stresses. These systems are complemented by thioredoxin and thioredoxin reductase (TR) systems for maintaining cellular redox homeostasis. A diverse array of sensors is used by Mycobacterium for monitoring its intracellular redox status. Upon sensing reductive stress, Mtb uses a flexible and robust metabolic system for its dissipation. Branched electron transport chain allows Mycobacterium to function with different terminal electron acceptors and modulate proton motive force to fulfill energy requirements under diverse scenarios. Interestingly, Mtb utilizes variations in the tricarboxylic cycle and a number of dehydrogenases to dissipate reductive stress. Upon prolonged exposure to reductive stress, Mtb utilizes biosynthesis of storage and virulence lipids as a dissipative mechanism. Critical Issues: The mechanisms utilized by Mycobacterium for sensing and tackling reductive stress are not well characterized. Future Directions: The precise role of thiol buffering and TR systems in neutralizing reductive stress is not well defined. Genetic systems that respond to metabolic reductive stress and thiol reductive stress need to be mapped. Genetic screens could aid in identification of such systems. Given that management of reductive stress is critical for both actively replicating and persister mycobacteria, an improved understanding of the mechanisms used by mycobacteria for dissipation of reductive stress may lead to identification of vulnerable choke points that could be targeted for killing Mtb in vivo.

Post-operative delirium associated with metabolic alterations following hemi-arthroplasty in older patients.

post-operative delirium (POD) is a common complication in older patients, though a possible link between metabolic changes and POD development has yet to be investigated. older patients with hip fracture who underwent hemi-arthroplasty were recruited, and delirious states were assessed for 3 days after surgery using the confusion assessment method-Chinese revision. Simultaneously, fasting blood samples were collected on the morning of surgery and on the first post-operative day. Ultimately, 244 older patients who met the inclusion and exclusion criteria were assessed. Blood samples from 60 patients with POD and 60 matched controls were analysed using metabolomics platforms. sixty patients (24.6%) developed POD. Principal component analysis scores plot and cross-validated scores plots from orthogonal partial least squares-discriminant analysis were implemented to visualise the differences in metabolites between the two groups before and after surgery (P < 0.05). Our data indicate that levels of ω3 and ω6 fatty acids were lower in the POD group than in the NPOD (non-POD) group both before and after surgery; tricarboxylic cycle intermediate levels were lower in the POD group than in the NPOD group, but glycolysis products were higher in the POD group than in the NPOD group after surgery. Furthermore, the branched-chain amino acid (BCAA)/aromatic amino acid ratio was lower in the POD group than in the NPOD group after surgery. metabolic abnormalities, including deficiencies in ω3 and ω6 fatty acids, perturbations in tricarboxylic cycle and oxidative stress and metabolic imbalances in BCAA and AAA might contribute to POD development.

Targeting the Mitochondrial Pyruvate Carrier for Neuroprotection.

The mitochondrial pyruvate carriers mediate pyruvate import into the mitochondria, which is key to the sustenance of the tricarboxylic cycle and oxidative phosphorylation. However, inhibition of mitochondria pyruvate carrier-mediated pyruvate transport was recently shown to be beneficial in experimental models of neurotoxicity pertaining to the context of Parkinson’s disease, and is also protective against excitotoxic neuronal death. These findings attested to the metabolic adaptability of neurons resulting from MPC inhibition, a phenomenon that has also been shown in other tissue types. In this short review, I discuss the mechanism and potential feasibility of mitochondrial pyruvate carrier inhibition as a neuroprotective strategy in neuronal injury and neurodegenerative diseases.

Impaired ketogenesis and increased acetyl-CoA oxidation promote hyperglycemia in human fatty liver.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, and potentially morbid, disease that affects one-third of the U.S. population. Normal liver safely accommodates lipid excess during fasting or carbohydrate restriction by increasing their oxidation to acetyl-CoA and ketones, yet lipid excess during NAFLD leads to hyperglycemia and, in some, steatohepatitis. To examine potential mechanisms, flux through pathways of hepatic oxidative metabolism and gluconeogenesis were studied using five simultaneous stable isotope tracers in ketotic (24-hour fast) individuals with a wide range of hepatic triglyceride contents (0-52%). Ketogenesis was progressively impaired as hepatic steatosis and glycemia worsened. Conversely, the alternative pathway for acetyl-CoA metabolism, oxidation in the tricarboxylic (TCA) cycle, was upregulated in NAFLD as ketone production diminished and positively correlated with rates of gluconeogenesis and plasma glucose concentrations. Increased respiration and energy generation that occurred in liver when β-oxidation and TCA cycle activity were coupled may explain these findings, inasmuch as oxygen consumption was higher during fatty liver and highly correlated with gluconeogenesis. These findings demonstrate that increased glucose production and hyperglycemia in NAFLD is not a consequence of acetyl-CoA production per se, but how acetyl-CoA is further metabolized in liver.

PF197 A NEW CLASSIFICATION OF ACUTE MYELOID LEUKEMIA BASED ON INTEGRATED GENOMICS AND METABOLOMICS

Background:An expanding body of evidence indicates that targeting cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML). Pyrimidine biosynthesis, oxidative phosphorylation, amino acid and arginine metabolism are among the most relevant actionable pathways (Sykes et al. Cell 2016; Molina et al. Nat Med 2018; Jones et al. Cancer Cell 2018; Mussai et al. Blood 2015). However, few studies identified specific metabolic vulnerabilites (Fenouille et al. Nat Med 2017; Gallipoli et al. Blood 2018) due to the lack of a comprehensive characterization of AML cell metabolism.Aims:The study aimed to stratify AML patients based on their metabolic landscape and integrate their genomic and metabolic profiles.Methods:Genomic data of AML patients were obtained by whole exome sequencing (n = 165) or targeted NGS (n = 18). Of them, 119 AML were evaluated for serum metabolites by nuclear magnetic resonance (NMR), along with 145 healthy donors. Intracellular metabolites of AML bone marrow cells (35 CD34+ and 15 CD33+CD14− samples) and controls (21 cord blood CD34+ and 21 peripheral blood CD33+ samples) were analyzed by mass spectrometry (Metabolon).Results:Unsupervised hierarchical clustering of AML based on the intracellular metabolic landscape clearly defined 3 clusters (C, Fig.A). C1 was characterized by high metabolite concentration, C2 by low metabolite levels and C3 by a more complex metabolic profile. By integrating genomic data into the metabolic classification we obtained a significant clusterization (p = 0.029). C1 was enriched for NPM1‐mutated (mut) AML (50%), C2 for cases with altered chromatin/splicing genes or inv(3) (37% and 13%, respectively), C3 for TP53‐mut/aneuploid AML (35%). Moreover, C3 included all core binding factor AML. By training a Gaussian Process classifier on NMR data, we obtained a significant separation of C1 and C3 also at serum level (Fig. B,C). In particular, NPM1‐mut AML displayed abundance of tricarboxylic cycle byproducts, while glutamine and creatinine were increased in NPM1‐wildtype (wt) cases (Fig.D,E). At intracellular level, NPM1‐mut AML were characterized by increased intermediates of purine and pyrimidine metabolism, in line with an active biosynthetic activity. Moreover, unsupervised NMR data analysis identified a clear separation (0:7 accuracy) between TP53‐wt and mut/deleted AML, the latter showing reduced threonine, alanine, glucose, lactate and glutamine serum concentration (Fig.F).At genomic level, 88% of patients carried at least one mutation in a metabolism‐related gene including enzymes (encoded by nuclear or mitchondrial DNA) and metabolic regulators, with lipid metabolism, cellular respiration (IDH1/2 mutations and oxidative phosphorylation with altered UQCRH, FASTKD3, ND and COX genes), metabolism of carbohydrates (e.g. glycosyltransferase GXYLT1, GDP‐mannose biosynthetic process involving PMM2), glucose (e.g. glycolysis‐related genes HK2, HK3, PKLR, BPGM) and nucleotides (e.g. AK2, AK5: nucleotide phosphate group transfer, NME7: synthesis of nucleoside triphosphates) being the pathways most frequently targeted by mutations.Summary/Conclusion:AML is characterized by a number of functional and genomic‐driven metabolic alterations. The integration of genomic and metabolic profiles provided a novel refined AML classification and suggested subtype‐specific metabolic targets, including nucleotide metabolic pathways and bioenergetics in NPM1‐mut and TP53‐mut/del AML, respectively.imageSupported by: EHA research fellowship award, AIRC, FP7‐NGS‐PTL, Fondazione del Monte.

Antihyperglycemic Potential of Back Tea Extract Attenuates Tricarboxylic AcidCycle Enzymes by Modulating Carbohydrate Metabolic Enzymes in Streptozotocin-Induced Diabetic Rats.

The present study was aimed to investigate the effect of black tea extract on blood glucose, plasma insulin, Hemoglobin, carbohydrate metabolic enzymes and tricarboxylic enzymes in streptozotocin (STZ) induced diabetic rats. Diabetes was induced in male albino Wistar rats by intraperitoneal administration of STZ (40mg/kgbwt). Black tea extract was administered to diabetic rats at a dose of 25, 50 and 100mg/kg body weight for 30days. The effects of black tea extract on glucose, insulin and HbA1c levels were analyzed to confirm the effective dose. Administration of black tea extract to diabetic rats was significantly decreased the level of glucose, glycated hemoglobin and increased the levels of insulin in a dose dependent manner. The black tea extracts at a dose of 100mg/kgbwt showed a highly significant effect compared to other two doses (25 and 50mg/kgbwt). The effect produced by black tea extract (100mg/kgbwt) was comparable to that of glibenclamide (5mg/kgbwt) a reference anti diabetic drug. Therefore, 100mg/kgbwt was fixed as an effective dose and used for further analyses. Black tea extract was administered to diabetic rats at a dose of 100mg/kgbwt for 30days reinstated the altered levels of the plasma glucose, insulin, hemoglobin, glycosylated hemoglobin, carbohydrate metabolizing enzymes and tricarboxylic cycle enzymes in diabetic rats. Black tea extract administered to diabetic rats at a dose of 100mg/kg bwt for 30days reinstated the altered levels of the plasma glucose, insulin, hemoglobin, glycosylated hemoglobin, carbohydrate metabolizing enzymes and tricarboxylic cycle enzymes in diabetic rats. The effect produced by black tea extract of all the biochemical parameters were comparable with glibenclamide-used as a reference drug.

Analysis of protein‐protein interactions between isoforms of malate dehydrogenase and citrate synthase

Sequential enzymes involved in a metabolic pathway are often found in specific and dynamic protein‐protein interactions. The assembly of proteins into this metabolon allow the efficient transfer of substrate between adjacent proteins increasing the throughput and decreasing the potential for side metabolic reactions to occur between competing enzymes. One such pairing is the malate dehydrogenase (MDH) and citrate synthase (CS). In the forward direction of the tricarboxylic cycle, the oxidation of malate to OAA catalyzed by MDH is thermodynamically unfavorable. Coupling and potentially direct shuttling of product‐substrate affords the cycle to continue. The interaction site between mitochondrial MDH and CS has been partially identified via computational docking and cross‐linking studies. However, the domains and key residues of interaction and their impact on the actions and affinity of various isoforms of MDH and CS have not been identified. Using two biophysical techniques, protein thermal melts and surface plasminogen resonance, we have found both mitochondrial and cytosolic human MDH interact with CS (mitochondrial 3 fold greater than cytosolic), while plant MDH shows no interaction. Traditional pulldowns were used in addition to biophysical techniques. Using molecular crowding agents, we have identified the affinity and stoichiometry of interactions. This will allow us to probe the structures for key residues of interaction in the future.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolomic investigations in cerebrospinal fluid of Parkinson's disease.

The underlying mechanisms of Parkinson´s disease are not completely revealed. Especially, early diagnostic biomarkers are lacking. To characterize early pathophysiological events, research is focusing on metabolomics. In this case-control study we investigated the metabolic profile of 31 Parkinson´s disease-patients in comparison to 95 neurologically healthy controls. The investigation of metabolites in CSF was performed by a 12 Tesla SolariX Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICR-MS). Multivariate statistical analysis sorted the most important biomarkers in relation to their ability to differentiate Parkinson versus control. The affected metabolites, their connection and their conversion pathways are described by means of network analysis. The metabolic profiling by FT-ICR-MS in CSF yielded in a good group separation, giving insights into the disease mechanisms. A total number of 243 metabolites showed an affected intensity in Parkinson´s disease, whereas 15 of these metabolites seem to be the main biological contributors. The network analysis showed a connection to the tricarboxylic cycle (TCA cycle) and therefore to mitochondrial dysfunction and increased oxidative stress within mitochondria. The metabolomic analysis of CSF in Parkinson´s disease showed an association to pathways which are involved in lipid/ fatty acid metabolism, energy metabolism, glutathione metabolism and mitochondrial dysfunction.

Alterations in arginine and energy metabolism, structural and signalling lipids in metastatic breast cancer in mice detected in plasma by targeted metabolomics and lipidomics

BackgroundThe early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis.MethodsMice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics.ResultsBased on primary tumour growth and pulmonary metastases, 1–2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3–4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate (“Warburg effect”). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins.ConclusionsThe early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.

In vitro proteomic analysis of methapyrilene toxicity in rat hepatocytes reveals effects on intermediary metabolism.

The antihistaminic drug methapyrilene was withdrawn from the market in 1979 because of hepatocarcinogenicity in rats. Since then, the drug has been used as a model hepatotoxin especially for transcriptomic analyses using material from in vivo studies. Much less transcriptomics data are available from in vitro studies, and no studies have investigated proteomic effects of methapyrilene in vitro. Thus, the present study was aimed to characterize the proteomic response of primary rat hepatocytes to methapyrilene, to broaden our knowledge on the molecular mechanisms of methapyrilene toxicity, and to compare the results of collagen sandwich-cultured hepatocytes to in vivo data. In vitro methapyrilene concentrations (0.39µM, 6.25µM, and 100µM) were chosen to cover an in vivo-relevant range. Based on published pharmacokinetic data they correspond to concentrations in portal vein blood for previously in vivo-tested doses of methapyrilene, up to a concentration showing slight cytotoxicity. Analysis of proteomic alterations by two-dimensional gel electrophoresis and mass-spectrometric protein identification demonstrated consistent and concentration-dependent effects of methapyrilene, in particular on mitochondrial proteins. Data suggest substantial deregulation of amino acid and ammonia metabolism and effects on mitochondrial energy supply pathways. The effects identified in vitro concur well with into previous in vivo observations. Several effects, for example, the influence of methapyrilene on S-adenosylmethionine metabolism, have not been described previously. The data suggest that already non-toxic concentrations of methapyrilene alter components of the intermediary metabolism, such as branched-chain amino acid metabolism, as well as urea and tricarboxylic cycle enzymes. In summary, data substantially add to our knowledge on molecular mechanisms of methapyrilene hepatotoxicity at the protein level.

Metabolomic analysis and biochemical changes in the urine and serum of streptozotocin-induced normal- and obese-diabetic rats.

Diabetes mellitus (DM) is a chronic disease that can affect metabolism of glucose and other metabolites. In this study, the normal- and obese-diabetic rats were compared to understand the diabetes disorders of type 1 and 2 diabetes mellitus. This was done by evaluating their urine metabolites using proton nuclear magnetic resonance (1H NMR)-based metabolomics and comparing with controls at different time points, considering the induction periods of obesity and diabetes. The biochemical parameters of the serum were also investigated. The obese-diabetic model was developed by feeding the rats a high-fat diet and inducing diabetic conditions with a low dose of streptozotocin (STZ) (25mg/kg bw). However, the normal rats were induced by a high dose of STZ (55mg/kg bw). A partial least squares discriminant analysis (PLS-DA) model showed the biomarkers of both DM types compared to control. The synthesis and degradation of ketone bodies, tricarboxylic (TCA) cycles, and amino acid pathways were the ones most involved in the variation with the highest impact. The diabetic groups also exhibited a noticeable increase in the plasma glucose level and lipid profile disorders compared to the control. There was also an increase in the plasma cholesterol and low-density lipoprotein (LDL) levels and a decline in the high-density lipoprotein (HDL) of diabetic rats. The normal-diabetic rats exhibited the highest effect of all parameters compared to the obese-diabetic rats in the advancement of the DM period. This finding can build a platform to understand the metabolic and biochemical complications of both types of DM and can generate ideas for finding targeted drugs.

Postoperative Delirium Development Associated with Metabolic Alterations Following Hemi-Arthroplasty in Elderly

Background: Postoperative delirium (POD) is a common and harmful complication following surgery in elderly and possible link between metabolic changes during perioperative period and POD development is yet to be investigated. Methods: A nested case-control study was performed through metabolomics to find out the metabolic abnormalities between patients with and without POD. Elderly patients with hip fracture who had hemi-arthroplasty were recruited and their delirious states were assessed for three days after surgery. Simultaneously, fasting blood samples were collected on the morning of surgery and on the first postoperative day. Ultimately, 244 elderly patients who met the inclusion and exclusion criteria completed our observations. The blood samples from 60 patients with POD and 60 matched controls were analyzed using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Findings: Sixty patients (24.6%) developed POD. Eighteen metabolites differed between the 2 groups before surgery, and twenty-three metabolites differed between the 2 groups after surgery. Similarly, thirty metabolites in POD group differed before and after surgery, and thirty-two metabolites in NPOD (Non-POD) group differed before and after surgery. After retrieved KEGG database, our data indicated that, in POD group, ω3 and ω6 fatty acids were lower than NPOD group at both before and after surgery, intermediates of tricarboxylic cycle were lower than NPOD group but products of glycolysis were higher than NPOD group after surgery, branched-chain amino acids (BCAAs) / aromatic amino acids (AAAs) ratio was lower than NPOD group after surgery. Interpretation: The metabolic abnormalities, including deficiencies of ω3 and ω6 fatty acids, abnormal oxygen metabolism, metabolic imbalances in aromatic and branched-chain amino acids, may potentially diminish the brain's inherent protective capacity and hence could contribute to the POD development. Funding Statement: This work was supported by grants from the Shanghai Municipal Commission of Health and Family Planning Foundation for Key Developing Disciplines (2015ZB0103) and the Shanghai Jiao Tong University Biomedical Engineering Cross Research Foundation (YG2015MS15). Declaration of Interests: The authors report no conflicts of interest. Ethics Approval Statement: The study was approved by the Ethics Committee of Shanghai Sixth People’s Hospital affiliated with Shanghai Jiao Tong University (Shanghai, China) and was registered (ChiCTR-CPC-15006141). Written informed consent was obtained from all the participants prior to the study.

Abstract B026: SNX-2112 interferes with mitochondrial metabolism in TP53 mutant tumors

Abstract Introduction: SNX-5422 is an orally active prodrug of SNX-2112, a potent, highly selective inhibitor of Hsp90 with epigenetic character that has shown antitumor activity in clinical trials. K562 cells have loss of p53 expression by nonsense-mediated mRNA decay. Since both loss of function TP53 mutations and MYC-driven tumors have been linked to aberrant cancer metabolism, including increased glycolysis, the effect of SNX-2112 on oncometabolic networks in the TP53 mutant chronic myelocytic leukemia K562 cell line was examined. Methods: The 50% inhibition concentration (IC50) of SNX-2112 on cell viability of cancer cell lines was determined using CellTiter-Glo® Luminescent Cell Viability Assay after incubation at various concentrations. All cells were cultured in media supplemented with 10% fetal bovine serum at 37°C, 5% CO2 and 95% humidity. Culture medium was purchased from GIBCO or Sigma, USA. Data from transcriptomics of the K562 cell line treated with SNX-2112 were obtained, and affected genes were analyzed for their roles in metabolic processes. Results: The IC50 values in TP53 null and in MYC-rearranged tumor cell lines obtained are shown in the table. Transcriptomics analysis of the effect of SNX-2112 on K562 onco-metabolism revealed that several genes involved in glycolysis (including AK2 and PCK2) and in the tricarboxylic cycle (e.g., DLST) were downregulated. SNX-2112 impaired the catabolism of branched-chain amino acids, as indicated by downregulation of BCAT, for example. Reductions were also observed in the following: ribosomal genes (MRPLs, MRPSs) associated with RNA translation; regulators of purine biosynthesis/one-carbon cycle (e.g., MTHFD1); and effectors of oxidative phosphorylation, including CYC1 and CYCS. Mitochondrial genes participating in carbohydrate (e.g., MECR, AUH) and protein metabolisms (MIPEP, SCO2), as well as redox and detoxification (e.g., SOD2, SUOX) pathways were downregulated. Conclusion: SNX-2112 demonstrated significant antitumor activity in TP53 null tumors and in rearranged MYC (8q24) hematologic and selected solid tumors (e.g., hepatocellular carcinoma, mesothelioma). This activity appears to be, in part, the result of interference with onco-metabolic pathways. Further research into this mechanism is ongoing to support this target in clinical trials. Cell LineTumor TypeIC50 (nM)TP53 null cell linesHep3BHepatocellular carcinoma7KATO IIIGastric carcinoma12NCI-H1299NSCLC - Adenocarcinoma8NCI-H358NSCLC - Adenocarcinoma10HL-60Acute promyelocytic leukemia31SK-MES-1NSCLC - SQCC10MEC-1Chronic lymphocytic leukemia5NCI-H520NSCLC - SQCC18RERF-LC-AINSCLC - SQCC54MYC-rearranged cell linesNAMALWABurkitt lymphoma4ST486Burkitt lymphoma4DaudiBurkitt lymphoma6RajiBurkitt lymphoma8CA46Burkitt lymphoma14SU-DHL-6DLBCL GCB - Double hit lymphoma6DOHH-2DLBCL GCB - Double hit lymphoma2WSU-DLCL-2DLBCL GCB - Richter’s FCL4NB-4Acute promyelocytic leukemia6AMO-1Multiple myeloma6MsTo 211HMesothelioma9NCI-H2452Mesothelioma7 Citation Format: Everardus Orlemans. SNX-2112 interferes with mitochondrial metabolism in TP53 mutant tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B026.

A molecular approach to drought-induced reduction in leaf CO2 exchange in drought-resistant Quercus ilex.

Drought-induced reduction of leaf gas exchange entails a complex regulation of the plant leaf metabolism. We used a combined molecular and physiological approach to understand leaf photosynthetic and respiratory responses of 2-year-old Quercus ilex seedlings to drought. Mild drought stress resulted in glucose accumulation while net photosynthetic CO2 uptake (Pn ) remained unchanged, suggesting a role of glucose in stress signaling and/or osmoregulation. Simple sugars and sugar alcohols increased throughout moderate-to-very severe drought stress conditions, in parallel to a progressive decline in Pn and the quantum efficiency of photosystem II; by contrast, minor changes occurred in respiration rates until drought stress was very severe. At very severe drought stress, 2-oxoglutarate dehydrogenase complex gene expression significantly decreased, and the abundance of most amino acids dramatically increased, especially that of proline and γ-aminobutyric acid (GABA) suggesting enhanced protection against oxidative damage and a reorganization of the tricarboxylic cycle acid cycle via the GABA shunt. Altogether, our results point to Q. ilex drought tolerance being linked to signaling and osmoregulation by hexoses during early stages of drought stress, and enhanced protection against oxidative damage by polyols and amino acids under severe drought stress.

Ketone-Based Metabolic Therapy: Is Increased NAD+ a Primary Mechanism?

The ketogenic diet’s (KD) anticonvulsant effects have been well-documented for nearly a century, including in randomized controlled trials. Some patients become seizure-free and some remain so after diet cessation. Many recent studies have explored its expanded therapeutic potential in diverse neurological disorders, yet no mechanism(s) of action have been established. The diet’s high fat, low carbohydrate composition reduces glucose utilization and promotes the production of ketone bodies. Ketone bodies are a more efficient energy source than glucose and improve mitochondrial function and biogenesis. Cellular energy production depends on the metabolic coenzyme nicotinamide adenine dinucleotide (NAD), a marker for mitochondrial and cellular health. Furthermore, NAD activates downstream signaling pathways (such as the sirtuin enzymes) associated with major benefits such as longevity and reduced inflammation; thus, increasing NAD is a coveted therapeutic endpoint. Based on differential NAD+ utilization during glucose- vs. ketone body-based acetyl-CoA generation for entry into the tricarboxylic cycle, we propose that a KD will increase the NAD+/NADH ratio. When rats were fed ad libitum KD, significant increases in hippocampal NAD+/NADH ratio and blood ketone bodies were detected already at 2 days and remained elevated at 3 weeks, indicating an early and persistent metabolic shift. Based on diverse published literature and these initial data we suggest that increased NAD during ketolytic metabolism may be a primary mechanism behind the beneficial effects of this metabolic therapy in a variety of brain disorders and in promoting health and longevity.