TCA Cycle Flux Research Articles

Abstract 280: Cardiac Deletion of the Mitochondrial Pyruvate Carrier Results in Dilated Cardiomyopathy with Preservation of Fatty Acid Catabolism

Pyruvate is an important metabolic substrate for the heart that is formed in the cytosol by glycolysis or conversion of lactate, and then must be transported into the mitochondrial matrix for further metabolism. The mitochondrial pyruvate carrier (MPC) is composed of MPC1 and MPC2 proteins that are each required for complex stability and transport activity. Indeed, mice with cardiac-specific knockout of MPC2 (CS-MPC2-/- mice) exhibited concomitant MPC1 degradation and marked reduction in pyruvate-stimulated mitochondrial respiration. While cardiac function and heart size was normal in 6 week old CS-MPC2-/- mice, serial echocardiograms demonstrated drastic increases in heart size, chamber dilation, and loss of contractile function at 10 and 16 weeks of age. Gene markers of heart failure, hypoxia, and fibrosis were markedly increased in CS-MPC2-/- hearts. Mitochondria isolated from 16 week old failing CS-MPC2-/- hearts exhibited normal respiration on glutamate/malate, succinate, palmitoylcarnitine, and 3-hydroxybutyrate/malate, indicating preservation of mitochondrial energetics with anaplerotic malate, or substrates to produce acetyl-CoA independent of pyruvate. Expression of genes encoding fat and ketone oxidation enzymes was not down-regulated in failing CS-MPC2-/- hearts as is typically observed in heart failure, suggesting these hearts may rely on fat or ketone body oxidation for ATP production. However, targeted metabolomics of hearts from 6 week old CS-MPC2-/- chow-fed mice suggested TCA cycle dysfunction due to decreased acetyl-CoA levels that are insufficient to condense with oxaloacetate, causing an accumulation of oxaloacetate/aspartate, malate, and fumarate. To determine whether increasing the availability of usable substrates (fatty acids and ketones) would rescue the cardiac dysfunction, CS-MPC2-/- mice were fed a high fat, low carbohydrate (ketogenic) diet. Ketogenic diet strikingly decreased hypertrophy and improved functional parameters in 10 week old mice. In conclusion, loss of mitochondrial pyruvate utilization leads to altered cardiac substrate metabolism and inability to maintain TCA cycle flux, resulting in dilated cardiomyopathy that can be corrected by administration of a ketogenic diet.

Metabolic profiles of exercise in patients with McArdle disease or mitochondrial myopathy

McArdle disease and mitochondrial myopathy impair muscle oxidative phosphorylation (OXPHOS) by distinct mechanisms: the former by restricting oxidative substrate availability caused by blocked glycogen breakdown, the latter because of intrinsic respiratory chain defects. We applied metabolic profiling to systematically interrogate these disorders at rest, when muscle symptoms are typically minimal, and with exercise, when symptoms of premature fatigue and potential muscle injury are unmasked. At rest, patients with mitochondrial disease exhibit elevated lactate and reduced uridine; in McArdle disease purine nucleotide metabolites, including xanthine, hypoxanthine, and inosine are elevated. During exercise, glycolytic intermediates, TCA cycle intermediates, and pantothenate expand dramatically in both mitochondrial disease and control subjects. In contrast, in McArdle disease, these metabolites remain unchanged from rest; but urea cycle intermediates are increased, likely attributable to increased ammonia production as a result of exaggerated purine degradation. Our results establish skeletal muscle glycogen as the source of TCA cycle expansion that normally accompanies exercise and imply that impaired TCA cycle flux is a central mechanism of restricted oxidative capacity in this disorder. Finally, we report that resting levels of long-chain triacylglycerols in mitochondrial myopathy correlate with the severity of OXPHOS dysfunction, as indicated by the level of impaired O2 extraction from arterial blood during peak exercise. Our integrated analysis of exercise and metabolism provides unique insights into the biochemical basis of these muscle oxidative defects, with potential implications for their clinical management.

Abstract 4971: IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors

Abstract Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production to support cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-010759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that AML, plus subsets of glioblastoma, neuroblastoma, lymphoma, melanoma, triple negative breast cancer (TNBC) and pancreatic cancer (PDAC) are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in several cancer types with IACS-010759 led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. Mechanistically, response to IACS-010759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, aspartate biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. Tumor growth inhibition and regression have been observed in molecularly defined subsets of TNBC and PDAC PDX xenograft models treated with IACS-010759, indicating that subsets of these indications are dependent on OXPHOS. Furthermore, treating TNBC or PDAC PDX models post-chemotherapy with IACS-010759 extends progression free survival, consistent with IACS-010759 targeting recently described metabolically adapted residual tumor cells. In orthotopic xenograft models of primary AML cells, daily oral treatment with 1-7.5 mg/kg IACS-010759 extended the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and a gene signature levels. Therefore, these readouts (OCR, aspartate and a nanostring geneset) have been validated for use as exploratory clinical biology of response endpoints. In parallel, completion of preclinical chemistry, manufacturing and control (CMC) as well as GLP safety and tolerability studies with IACS-010759 in multiple species have enabled the selection of a clinical entry dose. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, a Phase I clinical trial in relapsed, refractory AML was initiated in October 2016, with a parallel trial in solid tumors expected to initiate in early 2017. Initial results from the on-going AML trial will be disclosed. Citation Format: Jennifer Molina, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Jason Cross, Naval Daver, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Jing Han, Judy Hirst, Sha Huang, Yongying Jiang, Zhijun Kang, Marina Konopleva, Gang Liu, Helen Ma, Polina Matre, Timothy McAfoos, Funda Meric-Bernstam, Pietro Morlacchi, Florian Muller, Marina Protopopova, Melinda Smith, Sonal Sonal, Yuting Sun, Jay Theroff, Andrea Viale, Quanyun Xu, Carlo Toniatti, Giulio Draetta, Philip Jones, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4971. doi:10.1158/1538-7445.AM2017-4971

The TCA cycle as a bridge between oncometabolism and DNA transactions in cancer.

Cancer cells exploit metabolic rearrangements for sustaining their high proliferation rate and energy demand. The TCA cycle is a central metabolic hub necessary for ATP production and for providing precursors used in many biosynthetic pathways. Thus, dysregulation of the TCA cycle flux is frequently observed in cancer. The identification of mutations in several enzymes of the TCA cycle in human tumours demonstrated a direct connection between this metabolic pathway and cancer occurrence. Moreover, changes in the expression/activity of these enzymes were also shown to promote metabolic adaptation of cancer cells. In this review, the main genetic and non-genetic alterations of TCA cycle in cancer will be described. Particular attention will be given to extrametabolic roles of TCA cycle enzymes and metabolites underlying the regulation of nuclear and mitochondrial DNA transactions.

Metabolic impact of nutrient starvation in mevalonate-producing Escherichia coli

The aim of this work was to enhance mevalonate yield from glucose in Escherichia coli by essential nutrient starvations and to reveal these effects on the central carbon metabolism. Stationary phase culture without essential nutrients such as nitrogen, sulfur, and magnesium was evaluated using an engineered E. coli introducing mvaE and mvaS genes from Enterococcus faecalis. Sulfur starvation resulted in the highest mevalonate yield of 0.61C-molC-mol−1 from glucose. The metabolic impacts of nutrient starvation were investigated by 13C-metabolic flux analysis. Under nitrogen starvation, the flux of the TCA cycle was large, causing high CO2 production. This was caused by degradation of mevalonate synthesis pathway enzymes. Under magnesium starvation, NADPH production was decreased, which limited mevalonate synthesis and promoted an overflow of acetate. Sulfur starvation not only suppressed the TCA cycle flux, but also supplied NADPH for mevalonate synthesis.

Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli

Almost all synthetic pathways for biofuel production are designed to require endogenous metabolites in glycolysis, such as phosphoenolpyruvate, pyruvate, and acetyl-CoA. However, such metabolites are also required for bacterial cell growth. To reduce the metabolic imbalance between cell growth and target chemical production, we previously constructed a metabolic toggle switch (MTS) as a conditional flux redirection tool controlling metabolic flux of TCA cycle toward isopropanol production. This approach succeeded to improve the isopropanol production titer and yield while ensuring sufficient cell growth. However, excess accumulation of pyruvate, the precursor for acetyl-CoA synthesis, was also observed. In this study, for efficient conversation of pyruvate to acetyl-CoA (pyruvate oxidation), we designed a synthetic metabolic bypass composed of poxB and acs with the MTS for acetyl-CoA supply from the excess pyruvate. When this designed bypass was expressed at the appropriate expression level associated with the conditional metabolic flux redirection, pyruvate accumulation was prevented, and the isopropanol production titer and yield were improved. Final isopropanol production titer of strain harboring MTS with the synthetic metabolic bypass improved 4.4-fold compared with strain without metabolic flux regulation, and it was 1.3-fold higher than that of strain harboring the conventional MTS alone. Additionally, glucose consumption was also improved 1.7-fold compared with strain without metabolic flux regulation. On the other hand, introduction of the synthetic metabolic bypass alone showed no improvement in isopropanol production and glucose consumption. These results showed that the improvement in bio-production process caused by synergy between the MTS and the synthetic metabolic bypass.

Exo-MFA – A 13C metabolic flux analysis framework to dissect tumor microenvironment-secreted exosome contributions towards cancer cell metabolism

Dissecting the pleiotropic roles of tumor micro-environment (TME) on cancer progression has been brought to the foreground of research on cancer pathology. Extracellular vesicles such as exosomes, transport proteins, lipids, and nucleic acids, to mediate intercellular communication between TME components and have emerged as candidates for anti-cancer therapy. We previously reported that cancer-associated fibroblast (CAF) derived exosomes (CDEs) contain metabolites in their cargo that are utilized by cancer cells for central carbon metabolism and promote cancer growth. However, the metabolic fluxes involved in donor cells towards packaging of metabolites in extracellular vesicles and exosome-mediated metabolite flux upregulation in recipient cells are still not known. Here, we have developed a novel empirical and computational technique, exosome-mediated metabolic flux analysis (Exo-MFA) to quantify flow of cargo from source cells to recipient cells via vesicular transport. Our algorithm, which is based on 13C metabolic flux analysis, successfully predicts packaging fluxes to metabolite cargo in CAFs, dynamic changes in rate of exosome internalization by cancer cells, and flux of cargo release over time. We find that cancer cells internalize exosomes rapidly leading to depletion of extracellular exosomes within 24h. However, metabolite cargo significantly alters intracellular metabolism over the course of 24h by regulating glycolysis pathway fluxes via lactate supply. Furthermore, it can supply up to 35% of the TCA cycle fluxes by providing TCA intermediates and glutamine. Our algorithm will help gain insight into (i) metabolic interactions in multicellular systems (ii) biogenesis of extracellular vesicles and their differential packaging of cargo under changing environments, and (iii) regulation of cancer cell metabolism by its microenvironment.

Abstract PR01: IACS-010759 a novel inhibitor of oxidative phosphorylation advancing into first-in-human studies to exploit metabolic vulnerabilities

Abstract Tumor cells normally depend on both glycolysis and oxidative phosphorylation (OXPHOS) to provide the energy and macromolecule building blocks needed to enable continued tumor cell growth. Genetic or epigenetic inactivation of one of these two redundant pathways represents a metabolic vulnerability that should be susceptible to an inhibitor of the other pathway. We have identified multiple contexts where all or a subset of these tumors demonstrate a dependence on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent inhibitor of complex I of oxidative phosphorylation. In isolated mitochondria or permeabilized cells, ATP production or oxygen consumption is inhibited at single digit nM concentrations in the presence of malate/glutamate, but not succinate. More directly, IACS-10759 inhibits the conversion of NADH to NAD+ in an immunoprecipitated complex I assay at low nM concentrations. Importantly, IACS-10759 is orally bioavailable with excellent pharmacokinetics properties in preclinical species, and has an overall profile suitable for clinical development. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. In multiple PDX models of primary AML IACS-10759 treatment extends the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling revealed that the response to IACS-10759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust preclinical response in multiple model systems, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, clinical supplies manufactured, and a Phase I clinical trial in AML will be initiated during the second quarter of 2016. This abstract is also being presented as Poster B35. Citation Format: Philip Jones, M Emilia Di Francesco, Jennifer M. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher A. Bristow, Christopher L. Carroll, Ningping Feng, Jason P. Gay, Mary K. Geck Do, Jennifer M. Greer, Marina Konopleva, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda G. Smith, Sonal Fnu, Jay P. Theroff, Giulio Draetta, Giulio Draetta, Carlo Toniatti, Joseph R. Marszalek. IACS-010759 a novel inhibitor of oxidative phosphorylation advancing into first-in-human studies to exploit metabolic vulnerabilities. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr PR01.

The Important Role of the Citric Acid Cycle in Plants

The TCA cycle is a central metabolic hub necessary for ATP production and for providing precursors used in many biosynthetic pathways. The TCA cycle also plays a very important role in plants.Thus, dysregulation of the TCA cycle flux is frequently observed in plants.The identification of transgenic poplar in several enzymes of the TCA cycle in plants demonstrated a direct connection between this metabolic pathway and the influence to TCA cycle. In this review, the main genetic and non-genetic alterations of TCA cycle in poplar will be described.

Where does liver fat go? A possible molecular link between fatty liver and diabetes.

An elevation of fatty acid delivery amplifies the TCA cycle flux with a rise in anaplerosis/cataplerosis, leading to a proportional rise in oxidative stress and inflammation in liver.

Abstract 335: Title: IACS-010759 is a novel clinical candidate that targets AML cells by inducing a metabolic catastrophe through inhibition of oxidative phosphorylation

Abstract Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production leading to robust cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. In an orthotopic xenograft model of primary AML cells derived from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 treatment with 3 and 10 mg/kg orally using a 5 on/2 off schedule extended the median survival by greater than 2-fold. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling and flux analysis revealed that the response to IACS-10759 was associated with induction of a metabolic imbalance that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, and we expect to file an IND at the end of 1Q2016 and initiate a Phase I clinical trial in AML during the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Jing Han, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Gera, Jay Theroff, Quanyun Xu, Juliana Velez, Carlo Toniatti, Timothy Heffernan, Giulio Draetta, M. Emilia Di Francesco, Philip Jones, Joseph R. Marszalek. Title: IACS-010759 is a novel clinical candidate that targets AML cells by inducing a metabolic catastrophe through inhibition of oxidative phosphorylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 335.

Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled 13C6 glucose, 13C3 lactate, and 13C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.

(13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate–malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to 13C magnetic resonance spectroscopy (13C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable 13C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by 1H MRS and perhaps also by FDG-PET and hyperpolarized 13C MRS methods.

Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8% by inactivating competing pathways.

Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae.

The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.

PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth

Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.

Redistribution of metabolic fluxes in Chlorella protothecoides by variation of media nitrogen concentration

In this study, the Elementary Metabolite Unit (EMU) algorithm was employed to calculate intracellular fluxes for Chlorella protothecoides using previously generated growth and mass spec data. While the flux through glycolysis remained relatively constant, the pentose phosphate pathway (PPP) flux increased from 3% to 20% of the glucose uptake during nitrogen-limited growth. The TCA cycle flux decreased from 94% to 38% during nitrogen-limited growth while the flux of acetyl-CoA into lipids increased from 58% to 109% of the glucose uptake, increasing total lipid accumulation. Phosphoenolpyruvate carboxylase (PEPCase) activity was higher during nitrogen-sufficient growth. The glyoxylate shunt was found to be partially active in both cases, indicating the nutrient nature has an impact on flux distribution. It was found that the total NADPH supply within the cell remained almost constant under both conditions. In summary, algal cells substantially reorganize their metabolism during the switch from carbon-limited (nitrogen-sufficient) to nitrogen-limited (carbon-sufficient) growth.

Pseudo-transition Analysis Identifies the Key Regulators of Dynamic Metabolic Adaptations from Steady-State Data

Hundreds of molecular-level changes within central metabolism allow a cell to adapt to the changing environment. A primary challenge in cell physiology is to identify which of these molecular-level changes are active regulatory events. Here, we introduce pseudo-transition analysis, an approach that uses multiple steady-state observations of (13)C-resolved fluxes, metabolites, and transcripts to infer which regulatory events drive metabolic adaptations following environmental transitions. Pseudo-transition analysis recapitulates known biology and identifies an unexpectedly sparse, transition-dependent regulatory landscape: typically a handful of regulatory events drive adaptation between carbon sources, with transcription mainly regulating TCA cycle flux and reactants regulating EMP pathway flux. We verify these observations using time-resolved measurements of the diauxic shift, demonstrating that some dynamic transitions can be approximated as monotonic shifts between steady-state extremes. Overall, we show that pseudo-transition analysis can explore the vast regulatory landscape of dynamic transitions using relatively few steady-state data, thereby guiding time-consuming, hypothesis-driven molecular validations.

Hepatic Mitochondrial Pyruvate Carrier 1 Is Required for Efficient Regulation of Gluconeogenesis and Whole-Body Glucose Homeostasis

Gluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D.

Tracer-based assessments of hepatic anaplerotic and TCA cycle flux: practicality, stoichiometry, and hidden assumptions.

Two groups recently used different tracer methods to quantify liver-specific flux rates. The studies had a similar goal, i.e., to characterize mitochondrial oxidative function. These efforts could have a direct impact on our ability to understand metabolic abnormalities that affect the pathophysiology of fatty liver and allow us to examine mechanisms surrounding potential therapeutic interventions. Briefly, one method couples the continuous infusion of [(13)C]acetate with direct real-time measurements of [(13)C]glutamate labeling in liver; the other method administers [(13)C]propionate, in combination with other tracers, and subsequently measures the (13)C labeling of plasma glucose and/or acetaminophen-glucuronide. It appears that a controversy has arisen, since the respective methods yielded different estimates of the anaplerotic/TCA flux ratio (VANA:VTCA) in "control" subjects, i.e., the [(13)C]acetate- and [(13)C]propionate-derived VANA:VTCA flux ratios appear to be ∼1.4 and ∼5, respectively. While the deep expertise in the respective groups makes it somewhat trivial for each to perform the tracer studies, the data interpretation is inherently difficult. The current perspective was undertaken to examine potential factors that could account for or contribute to the apparent differences. Attention was directed toward 1) matters of practicality, 2) issues surrounding stoichiometry, and 3) hidden assumptions. We believe that the [(13)C]acetate method has certain weaknesses that limit its utility; in contrast, the [(13)C]propionate method likely yields a more correct answer. We hope our discussion will help clarify the differences in the recent reports. Presumably this will be of interest to investigators who are considering tracer-based studies of liver metabolism.