Analysis of glycolytic flux using 13C-labeled glucose confirmed that 10 μM IA reduced the appearance of labeled pyruvate and lactate by approximately 50%, while 50 μM IA reduced labeling by approximately 90% (Figure 4—figure supplement 2). To maintain metabolic homeostasis, cells utilize feedback loops at multiple levels in an integrated metabolic-signaling network. Presumably you would be able to extract these data from previous live-cell imaging experiments, therefore allowing you to revise the paper quickly. We now provide data from MCF12A and 184A1 (independently derived mammary epithelial cell lines) that show similar kinetics to MCF10A in response to the same perturbagens (Figure 3—figure supplement 2). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. Students rely on this text to help them quickly review, assimilate, and integrate large amounts of critical and complex information. – during glycolysis, ATP donates a phosphate group to glucose to form glucose 6-phosphate; Biological Oxidation and Reduction Half-Reactions. Write. Similar kinetics were observed at all reporter expression levels, with moderate trends toward higher pulse frequency and shorter pulse duration in cells with higher reporter expression (Figure 3—figure supplement 1). Curcumin supplementation for 12 weeks significantly increased AApoAII amyloid deposition relative to controls, especially in the liver and spleen. A histogram centered around 0 indicates a population in which there is no systematic trend in the correlation, while skew to the left or right indicates a negative or positive trend in correlation, respectively. Learn. The MCF10A full growth medium consisted of Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (#11330, Life Technologies, Carlsbad, CA), supplemented with 5% horse serum (Life Technologies), EGF (20 ng/ml), insulin (10 μg/ml), hydrocortisone (0.5 μg/ml), cholera toxin (100 ng/ml), and penicillin (50 U/ml) and streptomycin (50 μg/ml). The color of the dark meat comes from the iron present in the cytochromes involved in oxygen‐consuming respiration. Oligomycin-induced fluctuations in AMPK differed from IA-induced oscillations in their longer time scale (2–6 hr), in their irregular nature, and in that they were not tightly associated with coordinated changes in NADH and Akt indices (Figure 5—figure supplement 1A–B). We thank P Tsou and L Cantley for providing the AMPKAR plasmid; G Gao, J Locasale, and T Muranen for providing reagents; D Clapham, T Schwarz, V Mootha, M Vander Heiden, S Gaudet, I Harris, and members of the Albeck laboratory, Brugge laboratory, and the Yellen laboratory for their comments. At glucose concentrations of 3.4 mM and 1.7 mM, cells were unable to achieve full adaptation, with <25% and <10% of cells returning to baseline within 2 hr, and subsequent pulses in AMPK index were relatively dampened and prolonged. The AMPK and Akt pathways play key roles in this balance, intersecting through multiple crosstalk points and feedback loops to control both glucose metabolism (Figure 1—figure supplement 1) and protein translation at the level of mTOR. NADH index was calculated as a ratio of the background-subtracted nuclear T-Sapphire to YFP signal. glycolysis may offer a promising strategy to preferentially kill cancer cells. This allows the regulation of several pathways to be coordinated. Although O-GlcNAcylation can regulate many cell biological pathways, recent evidence suggests that it is a key regulator of metabolic pathways … catabolic reactions. It is unclear how much of the difference is due to training and how much to heredity. Prior to imaging, cells were placed in iGM lacking pyruvate and EGF and containing 2.5 mM glutamine, 3.4 mM glucose, and 10 μg/ml insulin. This occurs through substrate availability, … Cells cultured in combinations of glucose, pyruvate, or glutamine displayed varying elevated levels of steady-state AMPK index (Figure 1—figure supplement 2A–B), demonstrating that AMPKAR2 could monitor AMPK status across a range of physiological concentrations of nutrients in individual live cells. The warburg effect: how does it benefit cancer cells? All three probes reported predictable responses to major metabolic stressors, and some perturbations were accompanied by oscillatory responses in the metabolic/signaling state, many of which persisted for hours. Although all referees felt that the revision was markedly improved, they also felt that the issue of whether the sensors are influencing metabolism at the cellular level was not addressed thoroughly enough. (B) Single-cell measurements of AMPK index in the presence of EGF, insulin, or both, with exposure to 1.8 μg/ml oligomycin at time 0. To monitor Akt activity, we thus fused a red fluorescent protein mCherry to a truncated FOXO3a gene in which transcriptional activity was abrogated to minimize any interference on endogenous gene transcription, a strategy previously shown to specifically report Akt activity (Gross and Rotwein, 2016; Maryu et al., 2016). These … In the context of tumor microenvironments with fluctuating nutrient and oxygen supply, such instability is likely deleterious and may create selective pressure for genetic alterations to enhance glycolysis, such as activating mutations in the PI3K/Akt pathway, which are among the most frequent mutations across all cancer types. Second, 20 μl N-(tert-butyldimethylsilyl)-N- methyl-trifluoroacetamide with 1% tert-butyldimethylchlorosilane (TBDMS) (Sigma) was added and samples were incubated at 60°C for 1 hr. To track PI3K/Akt pathway activity, we constructed a reporter based on the Forkhead transcription factor FOXO3a. On the other hand, it has also been proposed that sustained glycolytic oscillations could play an important role in fine-tuning certain cellular functions, such as insulin secretion in pancreatic beta cells (Goodner et al., 1977). As expected, each of these compounds rapidly raised the mean AMPK index in a dose-dependent manner in cells cultured in growth medium, confirming that they perturb ATP homeostasis in proliferating MCF10A cells (Figure 3 and Videos 1–3). Oscillations of lactate released from islets of Langerhans: evidence for oscillatory glycolysis in beta-cells, Bioenergetic profile experiment using C2C12 myoblast cells. Growth factor (GF) stimulation activates the PI3K/Akt pathway, which plays a key role in proliferation by stimulating both cell cycle progression and mTOR activity, leading to increased protein translation. Unless noted otherwise, where single-cell recordings are shown, the displayed cells were chosen by random number generation in MATLAB with a threshold for minimum tracking time to eliminate cells in which recording was terminated prematurely due to failure of the tracking algorithm, and the chosen tracks were manually verified to be representative of successfully tracked cells and consistent with the overall range of cell behaviors. measuring conversion of 13C-glucose to 13C-lactate). ATP inhibits pyruvate kinase, similar to the inhibition of PFK. While this trend could suggest that reporter expression influences dynamics to a limited extent, the effect size is substantially weaker than the other effects we report in our study and is not strong enough to suggest that reporters severely distort the dynamics of the observed metabolic responses. For instance, glycolysis is regulated by feedback control at the level of phosphofructokinase, which senses the availability of ATP and the respiratory intermediate citrate. In the figure, the standard free energies and the concentrations were used to compute the total free energy differences between products and reactants at each step. (C) Quantification of pulsatile behavior in G0/G1 relative to S/G2. [U-13C6] glucose-containing medium was aspirated and cells were washed once with ice-cold saline, after which 500 μl of −20°C methanol was added to each well to quench metabolism. A two-part list of links to download the article, or parts of the article, in various formats. Should be able to break down all relevant reactions into their half-reactions; Helps determine the number of electrons that are being transferred The highly dynamic nature of AMPK responses at the single-cell level was unexpected, and we therefore investigated whether these effects were a consequence of the particular imaging methods or experimental systems used. We then used this framework to examine how metabolic adaptation functions in proliferating cells. Derivatized metabolite samples were analyzed on a 6890N GC with a DB-35ms Ultra Inert capillary column coupled to a 5975B Inert XL MS (Agilent, Santa Clara, CA). In the case of IA treatment, where a bottleneck is imposed between upper and lower glycolysis, higher glucose input stimulated by insulin would, by mass action, force increased flux through this bottleneck. In other circumstances, the rapid changes in ATP and other metabolites made possible by glycolysis may expose the cell to oscillatory behavior, as slower regulatory processes attempt to catch up. IA inhibits GAPDH, limiting both glycolysis and NADH production, yet the data suggest that glycolysis is required for oscillations in the NADH/NAD+ ratio. The result of this analysis is that the kinetics of oscillatory behavior are qualitatively similar across all reporter expression levels. Cells shown were manually selected to best represent the phase relationship visible in the majority of cells. Together, we think that these new analyses adequately establish that expression of reporters is effectively neutral for the effects being studied. To maintain metabolic homeostasis, cells utilize feedback loops at multiple levels in an integrated metabolic-signaling network. Hexokinase. For Peredox calibration using lactate and pyruvate, DMEM (Life Technologies #31053) with indicated lactate and pyruvate concentrations were made; cells were washed one to two times with the media prior to imaging. The net products of glycolysis are 2ATP and a reduced NAD (N icotinamide A denine D inucleotide) or NADH. However, there is a difficulty in comparing these experiments, as the bulk assay includes all of the subcellular pools of NADH and NAD+, while the reporter only indicates the cytosolic/nuclear pool. Imaging medium was iGM lacking pyruvate, with IA added at time 0. ATP is used as the energy carrier molecule and NADH is used to produce more ATP by oxidative phosphorylation. After cell tracking with the YFP images, the coordinates were applied to the other fluorescent channels. Altogether, these data suggest a model in which increased flux through glycolysis stimulated by insulin is able to attenuate recurrent ATP shortages during continued inhibition of oxidative phosphorylation (Figure 5—figure supplement 2). Statistical comparisons between samples are displayed using notches on the box plots; two samples in which notches do not overlap differ in their medians at the 5% significance level. Prior to imaging, cells were placed in iGM lacking pyruvate and glutamine at different glucose concentrations, and were treated at time 0 with 1.8 µg/ml oligomycin. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. To understand why cells fail to reach stable adaptation under some conditions, we focused first on the rapid oscillations triggered by IA treatment (Figure 4A). Therefore it is still possible that the sensors themselves contribute to the metabolic activities they were designed to report. The neutralization of this patch completely abolished recombinational DNA repair due to defects in the recruitment of Rad51 to DNA damage sites. Suppression was most potent with BEZ235, which inhibits PI3K, mTORC1, and mTORC2 activity, and strong but somewhat less potent with Torin1, which inhibits both mTORC1 and mTORC2. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. It is a general rule of metabolic regulation that pathways are regulated at the first committed step. AMP activates the reaction. The instrument was operated in electron ionization mode with an energy of 70 eV. CliffsNotes study guides are written by real teachers and professors, so no matter what you're studying, CliffsNotes can ease your homework headaches and help you score high on exams. The inlet temperature was held at 270°C. [Editors' note: further revisions were requested prior to acceptance, as described below.]. Typically, >500 individual cell recordings were scored for each condition and plotted using a box plot, with the median shown as a red line, 25th-75th percentile (the interquartile range) as the bottom and top of the box, respectively. In contrast, oligomycin induced an immediate increase in AMPK index that peaked at ~40 min but then fell, followed by a series of irregular pulses of AMPK activity ranging in duration from 1 to 3 hr (Figure 3B). The phase relationship between the measurable variables in this system indicates the existence of delays between feedback activation and recovery of ATP and NADH and supports a model in which the slowed flux through glycolysis due to IA treatment triggers a cyclic series of feedback events that drive regular oscillations (Figure 4—figure supplement 4). Distributions represent measurements of >500 cells under each condition. Your article has been favorably evaluated by Jonathan Cooper (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. MCF10A-AKT-KTR/AMPKAR2 cells were imaged in iGM2 as in Figure 4E, and were treated at time 0 with 10 µM IA. Think of the compounds in the free energy diagram as pools—where does a pool spill into a lower one, offering the possbility of control? Mn porphyrin regulation of aerobic glycolysis: implications on the activation of diabetogenic immune cells. The cytoplasmic CFP, YFP, and RFP signals were calculated as the mean pixel value within a cytoplasmic ‘donut’ mask, which consisted of an outer rim 3–4 μm from the nuclear mask and the inner rim as the perimeter of the eroded nuclear mask or 2–3 μm from the original nuclear mask. If glycolysis is activated, then the activity of pyruvate kinase must also be increased in order to allow overall carbon flow through the pathway. Pyruvate also had no effect on IA-induced AMPK index oscillations in the presence of glucose, although it rendered NADH index oscillations undetectable by lowering the resting NADH/NAD+ ratio (Figure 4—figure supplement 1D–E). Glycolysis is regulated by the concentration of glucose in the blood, the relative concentration of critical enzymes, the competition for the intermediate products of glycolysis and the levels of certain hormones in the bloodstream. Created by. N = 4, representative. (07) Several steps in glycolysis involve a transfer reaction, and some involve multiple processes. N = 4, combined. (E) Quantification of pulse score for each condition shown in (D). In live cells, AMPK activation was calculated based on a linear ratiometric method (Birtwistle et al., 2011), which we term ‘AMPK index’ (see Materials and methods for details of activity calculations). Pyruvate is one product of glycolysis, and can be shuttled into other … Remember that for any reaction, the free energy change depends on two factors: the free energy difference between the products and reactants in the standard state and the concentration of the products and reactants. The media used in each experiment (prior to the indicated additions) were as follows: C – iGM (imaging-modified growth medium; see Materials and methods); D – iGM lacking glucose; G – iGM2; H – iGM lacking glucose; K – iGM lacking insulin; L – iGM. Phosphorylation decreases synthesis, and dephosphorylation decreases breakdown. Similarly, in the presence of insulin, moderate inhibition of glycolysis with a dose of IA too low to independently stimulate oscillations resulted in amplification of oligomycin-induced AMPK pulses (Figure 5—figure supplement 1C). Reciprocal regulation occurs when the same molecule or treatment (phosphorylation, for example) has opposite effects on catabolic and anabolic pathways. Cell lines stably expressing biosensors were generated by retroviral or lentiviral infection, or by transfection with the PiggyBac transposase system, (Yusa et al., 2011), followed by puromycin (1–2 μg/ml) selection and expansion of single clones. In order to lower the amounts of extracellular pyruvate and facilitate measurements of cytosolic NADH-NAD+ redox (Hung and Yellen, 2014) in some experiments, we used an alternate imaging medium formulation, termed iGM2, which consists of 95% DMEM (Life Technologies #31053) and 5% DMEM/F12 (Life Technologies #11039), supplemented with 0.3% bovine serum albumin, EGF, insulin, hydrocortisone, cholera toxin, and pen-strep as above. One of the best regulated steps of glycolysis is the 6-phosphofructo-1-kinase (Pfk1)-catalyzed reaction, which converts fructose-6-phosphate (F6P) into fructose-1,6-bisphosphate (F1,6P2). 500 μl of assay medium was added to each well and cells were incubated in a 37°C incubator without CO2 for 1 hr to allow cell equilibration with assay medium. In the presence of glutamine, treatment with insulin strongly suppressed oligomycin-induced pulses in AMPKAR index, relative to non-GF treated cells (Figure 5B and C). (A,B) Single-cell measurements of AMPK index in MCF10A cells in the presence of EGF (20 ng/ml), insulin (10 μg/ml), or both. Because ATP plays a central role in providing energy for many essential cellular processes, even short lapses in availability can potentially compromise cellular function and viability; it is likely that evolution has selected for feedback kinetics that rapidly reverse any decrease in ATP to prevent levels from falling dangerously low. Regulation of glycogen synthesis and breakdown. O-GlcNAcylation: key regulator of glycolytic pathways Elevated O-GlcNAcylation is emerging as a general characteristic of most cancers. MCF10A-Peredox cells treated with lactate, or pyruvate in combination with iodoacetate (IA) as a glycolytic blockade, exhibited rapid (<3 min) changes to reach maximal and minimal sensor responses, respectively (Figure 1F and G), indicative of cytosolic NADH-NAD+ redox sensing. A control reporter with a mutation in the NADH-binding site (Y98D) predicted to abrogate NADH binding failed to respond to the same conditions (Figure 1—figure supplement 3). It is also worth noting an alternative explanation for this correlation, which is that the cell-specific factors leading to higher reporter expression in certain cells may also modify their metabolic behavior. To define a ‘pulse score’ for each cell, the amplitudes (difference between baseline and peak value) for all detected peaks were summed and normalized by the length of time the index was recorded. When energy is plentiful, the reaction is slowed down. For GF titration, cells were placed in EGF/insulin-deficient medium for 2 days prior to imaging with appropriate concentrations of EGF and insulin. Single-cell traces were generated using the automated software DCellIQ (Li et al., 2010), followed by manual verification using a custom MATLAB program (MathWorks, Natick, MA) to correct tracking errors, or using a custom MATLAB image processing pipeline (Sparta et al., 2015) using global optimization of cell tracks (Jaqaman et al., 2008). The tools developed here will be of use in detecting and analyzing similar oscillations in other cell types and conditions. Introduction to Glycolysis. We therefore next investigated the involvement of Akt by simultaneously monitoring both Akt and AMPK in dual reporter MCF10A-AMPKAR2/AKT-KTR cells. AMPKAR2-EV was constructed by modifying the linker between the CFP and YFP in AMPKAR (Tsou et al., 2011) with an expanded EEVEE linker (Komatsu et al., 2011), and replacing the CFP and YFP fluorophores with mTurquoise2 and YPet, respectively; a PiggyBAC transposase-mediated delivery system (Yusa et al., 2011) was used to minimize recombination between CFP and YFP. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. These data all indicate that at 10 μM IA where oscillations are strongest, glycolytic flux is intermediate, supporting the conclusion that this treatment slows but does not eliminate flux through glycolysis. N = 5, representative. We also show representative single-cell traces from the bottom-10 and top-10 percentiles of expression for each reporter. N = 3, representative. All differences indicated with an asterisk were significant at the p<0.05 level. Numbers of independent replicates are indicated in each figure legend as ‘N’; we define ‘independent replicate’ here as a complete, separate performance of a time lapse imaging experiment with similar culture and treatment conditions, beginning from the plating of cells from bulk culture on an imaging plate and occurring on different days from other replicates. This regulation reduces the rate of formation of glucose‐6‐phosphate. Simultaneously, Akt activity promotes glucose metabolism by stimulating the activity of hexokinase (Roberts et al., 2013) and phosphofructokinase (Novellasdemunt et al., 2013) and translocation of glucose transporters (Glut1 and Glut4) to the cell surface (Sano et al., 2003; Wieman et al., 2007), while PI3K enhances the activity of hexokinase, phosphofructokinase, and aldolase to increase glycolytic flux (Hu et al., 2016; Inoki et al., 2012; Inoki et al., 2003). (A) Expanded view of a representative region of oscillatory AMPK index for cells cultured in iGM and treated with 10 µM IA. PLAY. The data presented here suggest that the regulation of glycolysis by glucose availability and insulin/PI3K signaling can play both positive and negative roles in bioenergetic stability. We refer to this construct here as AKT-KTR; the Akt activity indicated by its cytosolic-to-nuclear fluorescence ratio is referred to as the ‘Akt index’ (Figure 1I). In contrast, mitochondrial oxidation of glucose in slow‐red muscle makes more ATP but the process takes longer. Examine the diagram of phosphoenolpyruvate becoming pyruvate and describe, in words, all the changes that occur during this step. In the seventh and tenth steps of glycolysis, ADP is phosphorylated at the level of the substrate into ATP. (A) Mean Akt index, measured by AKT-KTR, following stimulation with EGF or insulin. We speculate that, at concentrations where CCCP acts only as an ionophore, a new stable steady state is reached due to ATP consumption by the F0/F1 ATPase working in reverse and pumping protons to maintain the mitochondrial electrochemical gradient; when proton flow is blocked by oligomycin (or potentially by high doses of CCCP), AMPK kinetics are determined by other processes, which we investigate below. These polar metabolite extracts were then dried in a centrifugal evaporator and stored at −80°C until derivatization for GC-MS analysis. Similarly, NADH index fluctuations were increased with Akt, PI3K, mTOR, or dual PI3K/mTOR inhibitors (Figure 7C and D). Similar kinetic patterns were also observed in other mammary epithelial cell lines (184A1 and MCF12A; Figure 3—figure supplement 3A), indicating that they are not specific to the MCF10A cell line. Color scale bar at upper left indicates NADH index, with blue representing low values and green, yellow, red, and violet indicating successively higher values. Similar to PI3K inhibitors, we find that high concentrations of etomoxir dampen but do not eliminate oscillations (Figure 4—figure supplement 3). A central function of cellular metabolic regulation is to ensure an adequate supply of metabolites for bioenergetics and biosynthetic processes. In this study, we established a panel of proliferative epithelial cells expressing multiple fluorescent biosensors to enable detailed tracking of single-cell metabolic responses. A comparison with more therapeutically or physiologically relevant compounds such as metformin might be informative and would enhance the overall relevance of the paper to a broad audience. Overall oxidative phosphorylation values is shown relative to S/G2, respectively chapter 5: Bioenergetics and glycolysis Getting the out... 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