Krebs Cycle Activity Research Articles

Breast cancer cells utilize T3 to trigger proliferation through cellular Ca2+ modulation

High levels of thyroid hormones are linked to increased risk and advanced stages of breast cancer. Our previous work demonstrated that the biologically active triiodothyronine (T3) facilitates mitochondrial ATP production by upregulating Ca2+ handling proteins, thereby boosting mitochondrial Ca2+ uptake and Krebs cycle activity. In this study, different cell types were utilized to investigate whether T3 activates a Ca2+-induced signaling pathway to boost cancer cell proliferation. Using live-cell imaging, biochemical assays, and molecular profiling, differences in intracellular signaling among MCF7 and MDA-MB-468 breast cancer cells, non-cancerous breast cells hTERT-HME1, and PC3 prostate carcinoma cells, previously found to be insensitive to thyroid hormones in terms of proliferation, were investigated. Our findings revealed that T3 upregulates 1,4,5-trisphosphate receptor 3 via thyroid hormone receptor α. This boosts mitochondrial Ca2+ uptake, reduction equivalent yield, and mitochondrial ATP production, supporting the viability and proliferation of breast cancer cells without affecting non-cancerous hTERT-HME1 or PC3 prostate carcinoma cells. Understanding the interplay between T3 signaling, organellar interaction, and breast cancer metabolism could lead to targeted therapies that exploit cancer cell vulnerabilities. Our findings highlight T3 as a crucial regulator of cancer metabolism, reinforcing its potential as a therapeutic target in breast cancer.Graphical

Vitamin B5 supports MYC oncogenic metabolism and tumor progression in breast cancer

Tumors are intrinsically heterogeneous and it is well established that this directs their evolution, hinders their classification and frustrates therapy1–3. Consequently, spatially resolved omics-level analyses are gaining traction4–9. Despite considerable therapeutic interest, tumor metabolism has been lagging behind this development and there is a paucity of data regarding its spatial organization. To address this shortcoming, we set out to study the local metabolic effects of the oncogene c-MYC, a pleiotropic transcription factor that accumulates with tumor progression and influences metabolism10,11. Through correlative mass spectrometry imaging, we show that pantothenic acid (vitamin B5) associates with MYC-high areas within both human and murine mammary tumors, where its conversion to coenzyme A fuels Krebs cycle activity. Mechanistically, we show that this is accomplished by MYC-mediated upregulation of its multivitamin transporter SLC5A6. Notably, we show that SLC5A6 over-expression alone can induce increased cell growth and a shift toward biosynthesis, whereas conversely, dietary restriction of pantothenic acid leads to a reversal of many MYC-mediated metabolic changes and results in hampered tumor growth. Our work thus establishes the availability of vitamins and cofactors as a potential bottleneck in tumor progression, which can be exploited therapeutically. Overall, we show that a spatial understanding of local metabolism facilitates the identification of clinically relevant, tractable metabolic targets.

Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. Primary human macrophages activated by IFNγ (M(IFNγ)) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M(IFNγ)i) and phagocytic (M(IFNγ)p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Dietary Supplementation with Lauric Acid Improves Aerobic Endurance in Sedentary Mice via Enhancing Fat Mobilization and Glyconeogenesis

BackgroundLauric acid (LA), a major, natural, medium-chain fatty acid, is considered an efficient energy substrate for intense exercise and in patients with long-chain fatty acid β-oxidation disorders. However, few studies have focused on the role of LA in exercise performance and related glucolipid metabolism in vivo. ObjectivesWe aimed to investigate the effect of dietary supplementation with LA on exercise performance and related metabolic mechanisms. MethodsMale C57BL/6N mice (14 wk old) were fed a basal diet or a diet containing 1% LA, and a series of exercise tests, including a high-speed treadmill test, aerobic endurance exercises, a 4-limb hanging test, and acute aerobic exercises, were performed. ResultsDietary supplementation with 1.0% LA accelerated the recovery from fatigue after explosive exercise (P < 0.05) and improved aerobic endurance and muscle strength in sedentary mice (P = 0.039). Lauric acid intake not only changed muscle fatty acid profiles, including increases in C12:0 and n–6/n–3 PUFAs (P < 0.001) and reductions in C18:0, C20:4n–6, C22:6n–3, and n–3 PUFAs (P < 0.05) but also enhanced fat mobilization from adipose tissue and fatty acid oxidation in the liver, at least partly via the AMP-activated protein kinase–acetyl CoA carboxylase pathway (P < 0.05). Likewise, LA supplementation promoted liver glyconeogenesis and conserved muscular glycogen during acute aerobic exercise (P < 0.05), which was accompanied by an increase in the mitochondrial DNA copy number and Krebs cycle activity in skeletal muscle (P < 0.05). ConclusionsDietary supplemental LA serves as an efficient energy substrate for sedentary mice to improve aerobic exercise endurance and muscle strength through regulation of glucolipid metabolism. These findings imply that LA supplementation might be a promising nutritional strategy to improve aerobic exercise performance in sedentary people.

AMP-activated protein kinase activators have compound and concentration-specific effects on brain metabolism.

AMP-activated protein kinase (AMPK) is a key sensor of energy balance playing important roles in the balancing of anabolic and catabolic activities. The high energy demands of the brain and its limited capacity to store energy indicate that AMPK may play a significant role in brain metabolism. Here, we activated AMPK in guinea pig cortical tissue slices, both directly with A769662 and PF 06409577 and indirectly with AICAR and metformin. We studied the resultant metabolism of [1-13C]glucose and [1,2-13C]acetate using NMR spectroscopy. We found distinct activator concentration-dependent effects on metabolism, which ranged from decreased metabolic pool sizes at EC50 activator concentrations with no expected stimulation in glycolytic flux to increased aerobic glycolysis and decreased pyruvate metabolism with certain activators. Further, activation with direct versus indirect activators produced distinct metabolic outcomes at both low (EC50) and higher (EC50 × 10) concentrations. Specific direct activation of β1-containing AMPK isoforms with PF 06409577 resulted in increased Krebs cycle activity, restoring pyruvate metabolism while A769662 increased lactate and alanine production, as well as labelling of citrate and glutamine. These results reveal a complex metabolic response to AMPK activators in brain beyond increased aerobic glycolysis and indicate that further research is warranted into their concentration- and mechanism-dependent impact.

Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation

Studying the effects of terahertz (THz) radiation on the proteome of temperature-sensitive organisms is limited by a number of significant technical difficulties, one of which is maintaining an optimal temperature range to avoid thermal shock as much as possible. In the case of extremophilic species with an increased temperature tolerance, it is easier to isolate the effects of THz radiation directly. We studied the proteomic response to terahertz radiation of the thermophilic Geobacillus icigianus, persisting under wide temperature fluctuations with a 60 °C optimum. The experiments were performed with a terahertz free-electron laser (FEL) from the Siberian Center for Synchrotron and Terahertz Radiation, designed and employed by the Institute of Nuclear Physics of the SB of the RAS. A G. icigianus culture in LB medium was THz-irradiated for 15 min with 0.23 W/cm2 and 130 μm, using a specially designed cuvette. The life cycle of this bacterium proceeds under conditions of wide temperature and osmotic fluctuations, which makes its enzyme systems stress-resistant. The expression of several proteins was shown to change immediately after fifteen minutes of irradiation and after ten minutes of incubation at the end of exposure. The metabolic systems of electron transport, regulation of transcription and translation, cell growth and chemotaxis, synthesis of peptidoglycan, riboflavin, NADH, FAD and pyridoxal phosphate cofactors, Krebs cycle, ATP synthesis, chaperone and protease activity, and DNA repair, including methylated DNA, take part in the fast response to THz radiation. When the response developed after incubation, the systems of the cell's anti-stress defense, chemotaxis, and, partially, cell growth were restored, but the respiration and energy metabolism, biosynthesis of riboflavin, cofactors, peptidoglycan, and translation system components remained affected and the amino acid metabolism system was involved.

O-GlcNAcylation suppresses TRAP1 activity and promotes mitochondrial respiration.

The molecular chaperone TNF-receptor-associated protein-1 (TRAP1) controls mitochondrial respiration through regulation of Krebs cycle and electron transport chain activity. Post-translational modification (PTM) of TRAP1 regulates its activity, thereby controlling global metabolic flux. O-GlcNAcylation is one PTM that is known to impact mitochondrial metabolism, however the major effectors of this regulatory PTM remain inadequately resolved. Here we demonstrate that TRAP1-O-GlcNAcylation decreases TRAP1 ATPase activity, leading to increased mitochondrial metabolism. O-GlcNAcylation of TRAP1 occurs following mitochondrial import and provides critical regulatory feedback, as the impact of O-GlcNAcylation on mitochondrial metabolism shows TRAP1-dependence. Mechanistically, loss of TRAP1-O-GlcNAcylation decreased TRAP1 binding to ATP, and interaction with its client protein succinate dehydrogenase (SDHB). Taken together, TRAP1-O-GlcNAcylation serves to regulate mitochondrial metabolism by the reversible attenuation of TRAP1 chaperone activity.

Role of the cystathionine β-synthase / H2S pathway in the development of cellular metabolic dysfunction and pseudohypoxia in down syndrome

BackgroundOverexpression of the transsulfuration enzyme cystathionine-β-synthase (CBS), and overproduction of its product, hydrogen sulfide (H2S) are recognized as potential pathogenetic factors in Down syndrome (DS). The purpose of the study was to determine how the mitochondrial function and core metabolic pathways are affected by DS and how pharmacological inhibition of CBS affects these parameters. Methods8 human control and 8 human DS fibroblast cell lines have been subjected to bioenergetic and fluxomic and proteomic analysis with and without treatment with a pharmacological inhibitor of CBS. ResultsDS cells exhibited a significantly higher CBS expression than control cells, and produced more H2S. They also exhibited suppressed mitochondrial electron transport and oxygen consumption and suppressed Complex IV activity, impaired cell proliferation and increased ROS generation. Inhibition of H2S biosynthesis with aminooxyacetic acid reduced cellular H2S, improved cellular bioenergetics, attenuated ROS and improved proliferation. 13C glucose fluxomic analysis revealed that DS cells exhibit a suppression of the Krebs cycle activity with a compensatory increase in glycolysis. CBS inhibition restored the flux from glycolysis to the Krebs cycle and reactivated oxidative phosphorylation. Proteomic analysis revealed no CBS-dependent alterations in the expression level of the enzymes involved in glycolysis, oxidative phosphorylation and the pentose phosphate pathway. DS was associated with the dysregulation of several components of the autophagy network; CBS inhibition normalized several of these parameters. ConclusionsIncreased H2S generation in DS promotes pseudohypoxia and contributes to cellular metabolic dysfunction by causing a shift from oxidative phosphorylation to glycolysis.

Abstract 2537: Beta-glucan reprograms immunomodulatory metabolism in human macrophage and ex vivo in lung cancer tissues

Abstract We combined multiplex Stable Isotope-Resolved Metabolomics (mSIRM) with Reverse Phase Protein Array (RPPA) to map the time course changes of IM metabolic network and key protein regulators in four human donors’ MΦ’s in response to differential polarization and whole glucan particulates (WGP) treatments. We found consistent and plastic network responses to polarization durations and WGP treatments compared to those of the mouse counterparts. Consistent responses included enhanced 15N2-tryptophan catabolism to quinolinate, 2H2-glucose oxidation to ribose/ribulose-5-phosphate, and conversion of 13C5-glutamine to itaconate in response to pro-inflammatory (M1) versus anti-inflammatory (M2) stimuli. WGP robustly induced the buildup of 2H2-glucose-derived 6-phosphogluconate, -lactate, and -IMP, 13C5-glutamine-derived fructose-1,6-bisphosphate, and increased enrichment of 2H-labeled UDP-N-acetylglucosamine in M2-MΦ’s. However, the Krebs cycle activity was variably enhanced by M2 stimuli or WGP treatment. The consistent effects were related to increased release of proinflammatory IM effectors IL-1β/CXCL10/IFNγ/TNFα by M1-MΦ’s and enhanced release of IL-1β/TNFα to above M1-MΦ’s levels in WGP-treated M2-MΦ’s while boosting the latter in anti-inflammatory IL-10 release and maintenance of NAD+ synthesis. They were also related to lower phagocytosis in M1-MΦ’s and WGP-treated M2-MΦ’s versus M2-MΦ’s. Together with the expression changes of key protein regulators, we suggest enhanced tryptophan catabolism with blocked NAD+ and UTP synthesis to be key to the consistent changes in immune functions in response to M1 stimuli. Likewise, increased glucose utilization via glycolysis and the oxidative branch of the pentose phosphate pathway, and blockade of glutamine-fueled N-linked glycosylation could be linked to reversion of M2 to M1-type immune functions. Reprogrammed Krebs cycle and glutamine conversion to UTP occurred variably in WGP-treated ex vivo organotypic tissue cultures (OTCs) of human non-small cell lung cancer (NSCLC), which could reflect variable M1 repolarization of tumor associated MΦ’s. This in turn correlated with IL-1β/TNFα releases and compromised tumor status, making patient-derived OTCs a unique model for studying variable immunotherapeutic efficacy in cancer patients. In conclusion, consistent and variable IM metabolic responses were evident in four human donors’ MΦ’s. WGP repolarized some M2 to M1-type responses while boosting other M2-type responses. NSCLC OTCs from six patients showed variable M1 repolarization in response to WGP. Citation Format: Teresa W. M. Fan, Saeed Daneshmandi, Teresa A. Cassel, Mohammad B. Uddin, James Sledziona, Patrick T. Thompson, Penghui Lin, Richard M. Higashi, Andrew N. Lane. Beta-glucan reprograms immunomodulatory metabolism in human macrophage and ex vivo in lung cancer tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2537.

Electrophilic thymol isobutyrate from Inula nervosa Wall. (Xiaoheiyao) ameliorates steatosis in HepG2 cells via Nrf2 activation

• Electrophilic components from Xiaoheiyao are knocked out using magnetic GSH probe. • Electrophilic components have the potential to ameliorate steatosis in HepG2 cells. • 10-Isobutyryloxy-8,9-epoxythymol isobutyrate is discovered as a bioactive compound. • 10-Isobutyryloxy-8,9-epoxythymol isobutyrate improves steatosis via Nrf2 activation. Xiaoheiyao , the rhizomes of Inula nervosa Wall., is a culinary spice known to contain some electrophilic Nrf2 activators that could potentially intervene in nonalcoholic fatty liver disease (NAFLD). In this study, the electrophilic components from Xiaoheiyao were annotated by comprehensive cheminformatics analyses on UHPLC-MS/MS data after being knocked out with glutathione (GSH)-immobilized magnetic microspheres. Their intervention effects against hepatic steatosis were investigated in free fatty acid (FFA)-treated HepG2 cells. The electrophilic components in Xiaoheiyao were found to be the major bioactive components preventing hepatic steatosis in FFA-treated HepG2 cells. In particular, 10-isobutyryloxy-8,9-epoxythymol isobutyrate (XHY-1) improved hepatic lipid metabolism disorder, Krebs cycle activity, oxidative phosphorylation, and the cellular and mitochondrial redox status. Moreover, XHY-1 activates the Nrf2-ARE signaling pathway through the up-regulation of Nrf2 expression and promotion of Nrf2 nuclear translocation. Molecular docking further showed that XHY-1 could conjugate with Cys151 in the BTB domain of Keap1 through the epoxy group. In conclusion, the electrophilic components of Xiaoheiyao , especially XHY-1, attenuated hepatic steatosis in FFA-treated HepG2 cells through activation of the Nrf2-ARE signaling pathway.

Residual Complex I Activity Supports Glutamate Catabolism and mtSLP via Canonical Krebs Cycle Activity During Acute Anoxia Without OXPHOS

Residual Complex I Activity Supports Glutamate Catabolism and mtSLP via Canonical Krebs Cycle Activity During Acute Anoxia Without OXPHOS

Disruption of Glycolysis, TCA Cycle, Respiratory Chain, Calcium and Iron Homeostasis in Doxorubicin Induced Cardiomyopathy-An In-silico Approach

Doxorubicin is a well-known anthracycline antibiotic that is frequently used to treat a variety of malignancies. However, its clinical use is limited due to its adverse consequences, most notably cardiomyopathy. In the present work, we evaluated the molecular mechanisms behind the impairment of cardiac energetics in doxorubicin-induced cardiomyopathy. According to molecular docking, the interaction of doxorubicin with phosphofructokinase (PKF) and α-enolase is likely to negatively affect glycolysis. The interaction between doxorubicin with HMOX1 results in the accumulation of free iron. The free iron contributes to the heme-driven toxicity and the oxidizing environment that results in reactive oxygen species (ROS) production resulting from cell death. Additionally, the interaction of doxorubicin with HMOX1 impairs the availability of iron required for the Krebs cycle and ETC function. The interaction between doxorubicin and PINK1 results in a reduced membrane potential, which results in calcium accumulation. On the other hand, a lack of iron and calcium in the mitochondrial matrix results in ATP depletion, impairing the Krebs cycle activity. At the same time, the primary cause of doxorubicin-induced cardiomyopathy is cardiac energy metabolism. Thus, our work shows that doxorubicin impairs the activity of PFK, α-enolase, HMOX1, and PINK1, resulting in ATP production failure. As a result of changes in the heart energy metabolism, this ultimately leads to dilated cardiomyopathy caused by doxorubicin. Understanding the critical function of cardiac energy metabolism in doxorubicin-induced cardiomyopathy is critical for overcoming the obstacles that effectively limit the clinical effectiveness of this life-saving anti-cancer treatment.

Mechano-energetic aspects of Barth syndrome.

Energy-demanding organs like the heart are strongly dependent on oxidative phosphorylation in mitochondria. Oxidative phosphorylation is governed by the respiratory chain located in the inner mitochondrial membrane. The inner mitochondrial membrane is the only cellular membrane with significant amounts of the phospholipid cardiolipin, and cardiolipin was found to directly interact with a number of essential protein complexes, including respiratory chain complexes I to V. An inherited defect in the biogenesis of cardiolipin causes Barth syndrome, which is associated with cardiomyopathy, skeletal myopathy, neutropenia and growth retardation. Energy conversion is dependent on reducing equivalents, which are replenished by oxidative metabolism in the Krebs cycle. Cardiolipin deficiency in Barth syndrome also affects Krebs cycle activity, metabolite transport and mitochondrial morphology. During excitation-contraction coupling, calcium (Ca2+ ) released from the sarcoplasmic reticulum drives sarcomeric contraction. At the same time, Ca2+ influx into mitochondria drives the activation of Krebs cycle dehydrogenases and the regeneration of reducing equivalents. Reducing equivalents are essential not only for energy conversion, but also for maintaining a redox buffer, which is required to detoxify reactive oxygen species (ROS). Defects in CL may also affect Ca2+ uptake into mitochondria and thereby hamper energy supply and demand matching, but also detoxification of ROS. Here, we review the impact of cardiolipin deficiency on mitochondrial function in Barth syndrome and discuss potential therapeutic strategies.

Cell Energy Budget Platform for Multiparametric Assessment of Cell and Tissue Metabolism.

Specific bioenergetic signature reports on the current metabolic state of the cell, which may be affected by metabolic rearrangement, dysfunction or dysregulation of relevant signaling pathways, altered physiological condition or energy stress. A combined analysis of respiration , glycolytic flux, Krebs cycle activity, ATP levels, and total biomass allows informative initial assessment. Such simple, high-throughput, multiparametric methodology, called cell energy budget (CEB ) platform, is presented here and demonstrated with particular cell and tissue models. The CEB uses a commercial fluorescent lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA), a phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), a bioluminescent ATP kit, and an absorbance-based total protein assay. All the assays are performed on a standard multi-label reader. Using the same readouts, the CEB approach can be extended to more detailed mechanistic studies, by targeting specific pathways in cell bioenergetics and measuring other cellular parameters, such as NAD(P)H, Ca2+, mitochondrial pH, membrane potential, redox state, with conventional fluorescent or luminescent probes.

Aspartate aminotransferase and cardiovascular disease—a narrative review

: This narrative review summarized the existing knowledge on the association between aspartate aminotransferase (AST) and cardiovascular disease (CVD). AST plays a key role in the metabolism of amino acids, maintenance of NAD+/NADH ratio in cells, Krebs cycle activity, synthesis of purine/pyrimidine bases, urea and protein synthesis and gluconeogenesis. In humans, AST exists as two genetically and immunologically distinct isoenzymes: cytoplasmic AST and mitochondrial AST and both forms are found in circulation. AST activity is widely distributed across human tissues with the highest activity found in heart, liver, skeletal muscle, kidney and brain. An elevated AST activity may reflect tissue damage (plasma membrane disruption or apoptosis), plasma membrane bleb formation, increased tissue expression and macroenzymes (complexes of AST with plasma proteins). Serum AST activity is increased in patients with acute myocardial infarction in proportion with the extent of myocardial necrosis. Epidemiological evidence suggests the existence of an association between AST activity and CVD or mortality with positive, inverse or U (or J)-shaped association patterns. An elevated AST level not occurring in the setting of inflammatory liver disease may signify an increased cardiovascular risk related to nonalcoholic fatty liver disease (NAFLD), cardiometabolic risk factors (metabolic syndrome, abdominal obesity, insulin resistance and diabetes), chronic alcoholism and structural heart disease (myocardial infarction or congestive heart failure). A positive association between AST and CVD is more common in epidemiological studies in Asian population. Low AST levels may reflect increased cardiovascular risk related to vitamin B6 deficiency, advanced chronic kidney or liver diseases and inflammatory diseases. High and low AST levels have clinical meaning and both of them should be analyzed.

Combining lipoic acid to methylene blue reduces the Warburg effect in CHO cells: From TCA cycle activation to enhancing monoclonal antibody production.

The Warburg effect, a hallmark of cancer, has recently been identified as a metabolic limitation of Chinese Hamster Ovary (CHO) cells, the primary platform for the production of monoclonal antibodies (mAb). Metabolic engineering approaches, including genetic modifications and feeding strategies, have been attempted to impose the metabolic prevalence of respiration over aerobic glycolysis. Their main objective lies in decreasing lactate production while improving energy efficiency. Although yielding promising increases in productivity, such strategies require long development phases and alter entangled metabolic pathways which singular roles remain unclear. We propose to apply drugs used for the metabolic therapy of cancer to target the Warburg effect at different levels, on CHO cells. The use of α-lipoic acid, a pyruvate dehydrogenase activator, replenished the Krebs cycle through increased anaplerosis but resulted in mitochondrial saturation. The electron shuttle function of a second drug, methylene blue, enhanced the mitochondrial capacity. It pulled on anaplerotic pathways while reducing stress signals and resulted in a 24% increase of the maximum mAb production. Finally, the combination of both drugs proved to be promising for stimulating Krebs cycle activity and mitochondrial respiration. Therefore, drugs used in metabolic therapy are valuable candidates to understand and improve the metabolic limitations of CHO-based bioproduction.

GLP‐1 Induces a Shift in Primary Carbon Metabolites in Late‐fasted Northern Elephant Seals

Insulin resistance has been suggested to be an adaptive response to food deprivation since it promotes the preservation of glucose and oxidation of fat. However, the insulin resistance‐like conditions that fasting mammals experience may result from reduced pancreatic sensitivity to glucose/capacity to secrete insulin. Glucagon‐like peptide 1 (GLP‐1) is a gastrointestinal hormone that enhances postprandial glucose‐stimulated insulin secretion. To better assess the effects of GLP‐1 on the metabolism of primary carbon metabolites associated with late‐fasting induced insulin resistance, we dose‐dependently infused late‐fasted seals (naturally adapted to prolonged fasting) with low (10 pM/kg; n=3) or high (100 pM/kg; n=4) GLP‐1 immediately following a glucose bolus (0.5g/kg), using glucose without GLP‐1 as control (n=5). The plasma metabolome was measured from samples collected at 5 time points just prior to and during the infusions and areas under the curve (AUC) were calculated.The median AUC fold changes between the GLP‐1‐treated (low and high doses combined) and control group were analyzed using ChemRICH which is a chemical similarity enrichment analysis software for metabolomics datasets that uses medical subject headings and Tanimoto substructure chemical similarity coefficients to cluster metabolites into non‐overlapping chemical groups. Statistically significant p‐values for clusters were obtained by self‐contained Kolmogorov–Smirnov tests. ChemRich mapped 149 of the identified metabolites to 23 nonoverlapping chemical classes, of which 2 were found to be significantly different between the GLP‐1‐treated and control group (false discovery rate adjusted P value &lt; 0.05). Treatment with GLP‐1 infusions was associated with altered Kreb Cycle activity represented by increased levels of tricarboxylic acids, and reduced levels of carbocyclic acids (benzoic acid being a key compound). Hence, GLP‐1 infusion induced a shift in primary carbon metabolites in a model of fasting‐induced insulin resistance.Support or Funding InformationThis work was supported by grants: NIH NHLBI HL091767, NIH NHLBI HL091767‐S1, NIH NHLBI K02HL103787, NIH NHLBI R01HL09176, and NIH U24 DK097154.

Identifying strategies to target the metabolic flexibility of tumours.

Plasticity of cancer metabolism can be a major obstacle to efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that, while inhibition of both glutaminase isoforms (Gls1 and Gls2) in tumours considerably delays tumourigenesis, glutamine catabolism continues, owing to the action of amidotransferases. Synergistic inhibition of both glutaminases and compensatory amidotransferases is required to block glutamine catabolism and proliferation of mouse and human tumour cells in vitro and in vivo. Gls1 deletion is also compensated for by glycolysis. Thus, co-inhibition of Gls1 and hexokinase 2 significantly affects Krebs cycle activity and tumour formation. Finally, the inhibition of biosynthesis of either serine (Psat1-KO) or fatty acid (Fasn-KO) is compensated for by uptake of circulating nutrients, and dietary restriction of both serine and glycine or fatty acids synergistically suppresses tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate that either pharmacological or dietary targeting of metabolic compensatory mechanisms can improve therapeutic outcomes.

Altered glucose catabolism in the presynaptic and perisynaptic compartments of SOD1G93A mouse spinal cord and motor cortex indicates that mitochondria are the site of bioenergetic imbalance in ALS.

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disease that develops because of motor neuron death. Several mechanisms occur supporting neurodegeneration, including mitochondrial dysfunction. Recently, we demonstrated that the synaptosomes from the spinal cord of SOD1G93A mice, an in vitro model of presynapses, displayed impaired mitochondrial metabolism at early pre-symptomatic stages of the disease, whereas perisynaptic astrocyte particles, or gliosomes, were characterized by mild energy impairment only at symptomatic stages. This work aimed to understand whether mitochondrial impairment is a consequence of upstream metabolic damage. We analyzed the critical pathways involved in glucose catabolism at presynaptic and perisynaptic compartments. Spinal cord and motor cortex synaptosomes from SOD1G93A mice displayed high activity of hexokinase and phosphofructokinase, key glycolysis enzymes, and of citrate synthase and malate dehydrogenase, key Krebs cycle enzymes, but did not display high lactate dehydrogenase activity, the key enzyme in lactate fermentation. This enhancement was evident in the spinal cord from the early stages of the disease and in the motor cortex at only symptomatic stages. Conversely, an increase in glycolysis and lactate fermentation activity, but not Krebs cycle activity, was observed in gliosomes from the spinal cord and motor cortex of SOD1G93A mice although only at the symptomatic stages of the disease. The cited enzymatic activities were enhanced in spinal cord and motor cortex homogenates, paralleling the time-course of the effect observed in synaptosomes and gliosomes. The observed metabolic modifications might be considered an attempt to restore altered energetic balance and indicate that mitochondria represent the ultimate site of bioenergetic impairment.

IDH1 Mutation Enhances Catabolic Flexibility and Mitochondrial Dependencies to Favor Drug Resistance in Acute Myeloid Leukemia

Isocitrate dehydrogenases (IDH) are involved in redox control and central metabolism. We hypothesized that key metabolic fluxes are selectively reprogrammed to maintain biosynthetic homeostasis and lower drug responses in IDH mutant acute myeloid leukemia cells. Here we show that metabolic reprogramming initiated by IDH1 mutation leads to marked increases in glucose, glutamine and fatty acid catabolism that along with enhancement of wild-type IDH enzyme activity contribute to provision of α-KG required for 2-HG synthesis and to replenish Krebs cycle intermediates for biosynthetic reactions, oxygen consumption and ATP production. Mechanistically, this occurs through both methylation-driven CEBPα activation of FAO and reprogramming of systemic metabolic fluxes through other pathways that augment catabolic flexibility. Consequently, this catabolic flexibility enhances Krebs cycle and OxPHOS activities that are not necessarily rescued by IDH mutant inhibitors or 2-HG reduction. This renders IDH1 mutant cells more resistant to chemotherapeutics but more susceptible to mitochondrial inhibition. Our findings provide a scientific rationale for innovative combinatory targeted therapies to treat this subgroup of patients, especially those unresponsive to or relapsing from IDH mutant-specific inhibitors.