Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation ...stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.
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•Mitochondria produce acetyl-CoA from glutamine during MPC inhibition•Alanine synthesis is suppressed during MPC inhibition•MPC inhibition activates GDH to supply pools of TCA cycle intermediates•GDH supports cell survival during periods of MPC inhibition
Cancer cells import pyruvate into their mitochondria and convert it to acetyl-CoA for bioenergetics and biosynthesis. Yang et al. demonstrate that inhibition of mitochondrial pyruvate transport activates a compensatory pathway in which intramitochondrial glutamine metabolism produces pyruvate and acetyl-CoA. Inhibiting this compensatory pathway limits cell survival and tumor growth.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hyperpolarized magnetic resonance allows for noninvasive measurements of biochemical reactions in vivo. Although this technique provides a unique tool for assaying enzymatic activities in intact ...organs, the scope of its application is still elusive for the wider scientific community. The purpose of this review is to provide key principles and parameters to guide the researcher interested in adopting this technology to address a biochemical, biomedical, or medical issue. It is presented in the form of a compendium containing the underlying essential physical concepts as well as suggestions to help assess the potential of the technique within the framework of specific research environments. Explicit examples are used to illustrate the power as well as the limitations of hyperpolarized magnetic resonance.
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IJS, KILJ, NUK, PNG, UL, UM
Mitochondria are critical for respiration in all tissues; however, in liver, these organelles also accommodate high-capacity anaplerotic/cataplerotic pathways that are essential to gluconeogenesis ...and other biosynthetic activities. During nonalcoholic fatty liver disease (NAFLD), mitochondria also produce ROS that damage hepatocytes, trigger inflammation, and contribute to insulin resistance. Here, we provide several lines of evidence indicating that induction of biosynthesis through hepatic anaplerotic/cataplerotic pathways is energetically backed by elevated oxidative metabolism and hence contributes to oxidative stress and inflammation during NAFLD. First, in murine livers, elevation of fatty acid delivery not only induced oxidative metabolism, but also amplified anaplerosis/cataplerosis and caused a proportional rise in oxidative stress and inflammation. Second, loss of anaplerosis/cataplerosis via genetic knockdown of phosphoenolpyruvate carboxykinase 1 (Pck1) prevented fatty acid-induced rise in oxidative flux, oxidative stress, and inflammation. Flux appeared to be regulated by redox state, energy charge, and metabolite concentration, which may also amplify antioxidant pathways. Third, preventing elevated oxidative metabolism with metformin also normalized hepatic anaplerosis/cataplerosis and reduced markers of inflammation. Finally, independent histological grades in human NAFLD biopsies were proportional to oxidative flux. Thus, hepatic oxidative stress and inflammation are associated with elevated oxidative metabolism during an obesogenic diet, and this link may be provoked by increased work through anabolic pathways.
Metabolic suppression is a hallmark of animal dormancy that promotes overall energy savings. Some diapausing insects and some mammalian hibernators have regular cyclic patterns of substantial ...metabolic depression alternating with periodic arousal where metabolic rates increase dramatically. Previous studies, largely in mammalian hibernators, have shown that periodic arousal is driven by an increase in aerobic mitochondrial metabolism and that many molecules related to energy metabolism fluctuate predictably across periodic arousal cycles. However, it is still not clear how these rapid metabolic shifts are regulated. We first found that diapausing flesh fly pupae primarily use anaerobic glycolysis during metabolic depression but engage in aerobic respiration through the tricarboxylic acid cycle during periodic arousal. Diapausing pupae also clear anaerobic by-products and regenerate many metabolic intermediates depleted in metabolic depression during arousal, consistent with patterns in mammalian hibernators. We found that decreased levels of reactive oxygen species (ROS) induced metabolic arousal and elevated ROS extended the duration of metabolic depression. Our data suggest ROS regulates the timing of metabolic arousal by changing the activity of two critical metabolic enzymes, pyruvate dehydrogenase and carnitine palmitoyltransferase I by modulating the levels of hypoxia inducible transcription factor (HIF) and phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Our study shows that ROS signaling regulates periodic arousal in our insect diapasue system, suggesting the possible importance ROS for regulating other types of of metabolic cycles in dormancy as well.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Increased glucose uptake and aerobic glycolysis are striking features of many cancers. These features have led to many techniques for screening and diagnosis, but many are expensive, less feasible or ...have harmful side-effects. Here, we report a sensitive
H/
H NMR method to measure the kinetics of lactate isotopomer and HDO production using a deuterated tracer. To test this hypothesis, HUH-7 hepatocellular carcinoma and AML12 normal hepatocytes were incubated with
H
glucose.
H/
H NMR data were recorded for cell media as a function of incubation time. The efflux rate of lactate-CH
, lactate-CH
D and lactate-CHD
was calculated as 0.0033, 0.0071, and 0.0.012 µmol/10
cells/min respectively. Differential production of lactate isotopomers was due to deuterium loss during glycolysis. Glucose uptake and HDO production by HUH-7 cells showed a strong correlation, indicating that monitoring the HDO production could be a diagnostic feature in cancers. Deuterium mass balance of
H
glucose uptake to
H-lactate and HDO production is quantitatively matched, suggesting increasing HDO signal could be used to diagnose Warburg (cancer) metabolism. Measuring the kinetics of lactate isotopomer and HDO production by
H and
H MR respectively are highly sensitive. Increased T
of
H-lactate isotopomers indicates inversion/saturation recovery methods may be a simple means of generating metabolism-based contrast.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Epigenetic gene regulation and metabolism are highly intertwined, yet little is known about whether altered epigenetics influence cellular metabolism during cancer progression. Here, we show that ...EZH2 and NRAS
mutations cooperatively induce progression of myeloproliferative neoplasms to highly penetrant, transplantable, and lethal myeloid leukemias in mice. EZH1, an EZH2 homolog, is indispensable for EZH2-deficient leukemia-initiating cells and constitutes an epigenetic vulnerability. BCAT1, which catalyzes the reversible transamination of branched-chain amino acids (BCAA), is repressed by EZH2 in normal hematopoiesis and aberrantly activated in EZH2-deficient myeloid neoplasms in mice and humans. BCAT1 reactivation cooperates with NRAS
to sustain intracellular BCAA pools, resulting in enhanced mTOR signaling in EZH2-deficient leukemia cells. Genetic and pharmacologic inhibition of BCAT1 selectively impairs EZH2-deficient leukemia-initiating cells and constitutes a metabolic vulnerability. Hence, epigenetic alterations rewire intracellular metabolism during leukemic transformation, causing epigenetic and metabolic vulnerabilities in cancer-initiating cells. SIGNIFICANCE: EZH2 inactivation and oncogenic NRAS cooperate to induce leukemic transformation of myeloproliferative neoplasms by activating BCAT1 to enhance BCAA metabolism and mTOR signaling. We uncover a mechanism by which epigenetic alterations rewire metabolism during cancer progression, causing epigenetic and metabolic liabilities in cancer-initiating cells that may be exploited as potential therapeutics.
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Metabolic flux analysis (MFA) combines experimental measurements and computational modeling to determine biochemical reaction rates in live biological systems. Advancements in analytical ...instrumentation, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), have facilitated chemical separation and quantification of isotopically enriched metabolites. However, no software packages have been previously described that can integrate isotopomer measurements from both MS and NMR analytical platforms and have the flexibility to estimate metabolic fluxes from either isotopic steady-state or dynamic labeling experiments. By applying physiologically relevant cardiac and hepatic metabolic models to assess NMR isotopomer measurements, we herein test and validate new modeling capabilities of our enhanced flux analysis software tool, INCA 2.0. We demonstrate that INCA 2.0 can simulate and regress steady-state 13C NMR datasets from perfused hearts with an accuracy comparable to other established flux assessment tools. Furthermore, by simulating the infusion of three different 13C acetate tracers, we show that MFA based on dynamic 13C NMR measurements can more precisely resolve cardiac fluxes compared to isotopically steady-state flux analysis. Finally, we show that estimation of hepatic fluxes using combined 13C NMR and MS datasets improves the precision of estimated fluxes by up to 50%. Overall, our results illustrate how the recently added NMR data modeling capabilities of INCA 2.0 can enable entirely new experimental designs that lead to improved flux resolution and can be applied to a wide range of biological systems and measurement time courses.
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•INCA 2.0 can model both MS and NMR isotopomer measurements.•INCA 2.0 can estimate fluxes from steady-state or dynamic labeling experiments.•Dynamic NMR measurements improve flux resolution compared to steady-state.•Integration of MS and NMR datasets improves precision of hepatic fluxes.•INCA 2.0 automates the analysis of sophisticated metabolic tracer experiments.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Purpose
To determine whether deuterated water (HDO) generated from the metabolism of 2H7glucose is a sensitive biomarker of cerebral glycolysis and oxidative flux.
Methods
A bolus of 2H7glucose was ...injected through the tail vein at 1.95 g/kg into Sprague‐Dawley rats. A 2H surface coil was placed on top of the head to record 2H spectra of the brain every 1.3 minutes to measure glucose uptake and metabolism to HDO, lactate, and glutamate/glutamine. A two‐point Dixon method based on a gradient‐echo sequence was used to reconstruct deuterated glucose and water (HDO) images selectively.
Results
The background HDO signal could be detected and imaged before glucose injection. The 2H NMR spectra showed arrival of 2H7glucose and its metabolism in a time‐dependent manner. A ratio of the HDO to glutamate/glutamine resonances demonstrates a pseudo–steady state following injection, in which cerebral metabolism dominates wash‐in of HDO generated by peripheral metabolism. Brain spectroscopy reveals that HDO generation is linear with lactate and glutamate/glutamine appearance in the appropriate pseudo–steady state window. Selective imaging of HDO and glucose is easily accomplished using a gradient‐echo method.
Conclusion
Metabolic imaging of HDO, as a marker of glucose, lactate, and glutamate/glutamine metabolism, has been shown here for the first time. Cerebral glucose metabolism can be assessed efficiently using a standard gradient‐echo sequence that provides superior in‐plane resolution compared with CSI‐based techniques.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Non‐alcoholic fatty liver disease (NAFLD) is characterized by >5% hepatic fat by mass. As one of the most common liver diseases, with a prevalence of roughly 24% in the U.S. adult population, NAFLD ...related morbidities account for a striking ~300 billion dollars a year in healthcare costs for the U.S. alone Shetty, A., & Syn, W. K. Federal practitioner: for the health care professionals of the VA, DoD, and PHS, (2019), 36(1), 14–19. NAFLD, both in humans and murine models of the disease, has been associated with increased rates of hepatic β‐oxidation and reactive oxygen species production Sunny, N. E., Parks, E. J., Browning, J. D., & Burgess, S. C. Cell metabolism, (2011). 14(6), 804–810. These characteristics are thought to drive a pro‐inflammatory state that causes hepatic damage, and can transition to the much more serious malady nonalcoholic steatohepatitis. To directly quantify hepatic β‐oxidation as a potential biomarker of NAFLD disease development and progression, we are developing deuterium magnetic resonance (DMR) techniques to monitor oxidation of D15‐octanoate. In the perfused mouse liver, we demonstrate that DMR measures of HDO production, as a direct byproduct of β‐oxidation, estimate hepatic β‐oxidation rates within 5% of standard quantitation by mass spectrometry of fatty acid depletion from perfusate. In a dietary model of NAFLD (60% fat by kcal, HFD) we observed increased overall b‐oxidation, but when normalized to the total liver weight, the HFD liver oxidized less than the low‐fat diet control. By coupling DMR spectroscopy, gas chromatography‐mass spectrometry and metabolic modelling, we were able to identify increases in total ketogenesis and FADH2 oxidation in high fat diet perfused livers. When normalizing metabolic flux to liver protein, both octanoate and FADH2 oxidation were significantly lower in high fat diet livers. Increased total fatty acid oxidation and ketogenesis implicate activation of compensatory mechanisms by the liver during NAFLD to address higher levels of circulating lipids. Decreases in efficiency/relative oxidation indicate an accrual of hepatic damage that hinders the efficient clearance of excess lipids. These results demonstrate the utility of DMR as a sensitive measure for detecting changes in metabolic activity during NAFLD progression, and suggests that a clinical imaging paradigm might be feasible.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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