Lactate secretion was reduced moderately by 10 μM IA and more strongly by 50 μM IA; together these measures indicate that glycolytic flux is reduced to an intermediate rate by 10 μM IA. 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. Oligomycin and IA-treated cells showed significantly increased pulse scores relative to untreated cells (Figure 3D). For the glycolytic stress test, growth medium was completely removed 24 hr after plating and replaced with glycolytic assay medium (XF base medium supplied with 5 mM glucose, 1 mM pyruvate, and 2 mM glutamine; pH 7.4) as described earlier. Regulation of glycolysis Three regulatory enzymes: Hexokinase & glucokinase Phosphofructokinase Pyruvate kinase Catalysing the irreversible reactions regulate glycolysis. Firstly glucose enters the cell via facilitated diffusion and is activated to glucose-6-phosphate, by ATP. While 1 µM CCCP induced greater average AMPK index than oligomycin, neither cell cycle progression nor cell viability were altered. High sugar levels stimulate the pancreas to produce insulin, which enhances the entry of glucose into the cell and increases the production of the critical glycolysis enzymes. Using a dataset of >3,000 cells we have divided the cells into bins by expression of either AMPKAR2 or Akt-KTR, and have calculated the distribution of pulse parameters within each of these subpopulations. We therefore next investigated the involvement of Akt by simultaneously monitoring both Akt and AMPK in dual reporter MCF10A-AMPKAR2/AKT-KTR cells. The most important regulatory step of glycolysis is the phosphofructokinase reaction. The pulse score for each cell thus increases with both the frequency and amplitude of peaks; examples of peak detection and corresponding scores are shown in Figure 3—figure supplement 1. However, optimization for such rapid and efficient adaptation comes with the potential that for certain conditions stable adaptation cannot be achieved, and unstable (e.g. At intermediate concentrations (5–10 μM), IA-induced oscillations persisted for as many as 20 hr but typically ended with cell death (Video 4). The data support the conclusion that these rapid oscillatory dynamics originate from changes in flux in glycolysis, with downstream metabolic processes playing little role. Whiskers indicate the range of values falling within 1.5 times the interquartile range outside of the interquartile range; outliers beyond this range are not shown. Reagents were from Sigma (St. Louis, MO) unless noted. Why fructose‐1,6‐bisphosphate? 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). We verified the sensing capability of AMPKAR2 by treating MCF10A-AMPKAR2 cells with the direct AMPK activator AICAR (Figure 1B and C). Spell. Because only two ATP molecules are produced per glucose consumed by glycolysis, a limited amount of energy is available for muscle activity. For experiments with drug addition, cells were placed in imaging medium until the addition of drug diluted as a 5x-20x concentrated spike. Bioenergetics & Regulation of Glycolysis. We hypothesized that IA-induced oscillations in AMPK activity and NADH/NAD+ ratio resulted from oscillations in glycolytic flux, triggered by partial inhibition of GAPDH activity by 10 μM IA and the resulting feedback-driven increases in the entry of glucose into glycolysis. For GF titration, cells were placed in EGF/insulin-deficient medium for 2 days prior to imaging with appropriate concentrations of EGF and insulin. Thus, cellular responses to metabolic perturbations do not correlate with overall magnitude of bioenergetic stress as measured by AMPKAR2. Slow‐red muscle doesn't work as quickly as fast‐white muscle but can be active for a longer period of time. Our results point to a central role for insulin/PI3K/Akt regulation of glycolysis in modulating cellular metabolic stability. We propose that this patch is a fundamental motif that facilitates interactions with auxiliary factors and is therefore essential for recombinational DNA repair. After background subtraction, DCellIQ or the MATLAB pipeline were used for image segmentation and tracking to determine nuclear masks based either on nuclear-localized Peredox-mCitrine or on the absence of YPet nuclear fluorescence of cytoplasmic-localized AMPKAR2. Quickly memorize the terms, phrases and much more. 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. Similar to PI3K inhibitors, we find that high concentrations of etomoxir dampen but do not eliminate oscillations (Figure 4—figure supplement 3). Statistical significance was determined as described in the Materials and methods section. We also now include multiple experiments, including radioisotope tracing of glycolytic products, quantifying the glycolytic flux under high IA, low IA, and untreated conditions (Figure 4—figure supplement 2). To maintain metabolic homeostasis, cells utilize feedback loops at multiple levels in an integrated metabolic-signaling network. Since this is after glucose had been split into two three-carbon molecules, each molecule of glucose results in four ATP produced. Oscillations in metabolite concentrations during the operation of the cycle in muscle extracts, Reviewing Editor; UT Southwestern Medical Center, United States, (via ORCID - An ORCID is a persistent digital identifier for researchers), Brigham and Women’s Hospital, United States, Massachusetts Institute of Technology, United States, Open annotations. The balance of glycogen synthesis (storing glucose) and breakdown (releasing glucose) is regulated by activation and inhibition of kinase (phosphorylates) and phosphatase (dephosphorylates) enzyme activity. The conditions where we observe unstable behavior – including inhibition of lower glycolysis or mitochondrial ATP production, or culture in the complete absence of insulin-stimulated glucose uptake – likely represent situations that are unusual under normal physiological function. (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. Curcumin is a polyphenol compound that exhibits multiple physiological activities. Clearly the oscillations are most dramatic under very harsh conditions (IA, oligomycin, etc.). 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. On the other hand, the rate of water flow can be controlled effectively at any point where one pool spills into a lower one. In contrast, flight muscle (a fast‐white muscle) contains few mitochondria; glucose is broken down largely by glycolysis. (C) Quantification of pulse score for each condition shown in (B). In 17 mM glucose (the baseline concentration for MCF10A media), oligomycin induced a rapid initial pulse of AMPK activity and subsequent adaptation, with >75% of cells returning to baseline AMPK index within 2 hr. However, feedback creates the potential for unstable network responses. N = 5, representative. We then compared the pulse scores for IA-treated cells in the presence of varying extracellular concentrations of glucose and pyruvate. GFs were added at time 0. Additional annotation was performed in ImageJ 2.0 (Fiji). Importantly, the large cell-to-cell variation in pACC staining pattern observed by immunofluorescence in cells fixed at later time points is exactly what would be expected given the asynchronous fluctuations observed in live-cell experiments. Our data also suggest that metabolic oscillations may be a wider phenomenon than previously thought, as we demonstrate their occurrence in cells not typically considered highly metabolically active, and also find that they may occur with heterogeneous phasing that makes oscillations impossible to detect without single cell methods. Time-lapse wide-field microscopy was performed as previously described (Hung et al., 2011; Albeck et al., 2013). When a cell has a higher concentration of ATP than ADP (i.e. (C) Quantification of pulsatile behavior in G0/G1 relative to S/G2. BEZ235 (Axon Medchem, Reston, VA), Torin-1 (Tocris Bioscience, Bristol, UK), Torin-2 (Selleck, Houston, TX), BKM120 (Axon Medchem), GDC0941 (Axon Medchem), Gefitinib (Axon Medchem), MK2206 (Selleck), PD 0325901 (Sigma and Selleck), and Rad001 (SU2C PI3K Dream Team Mouse Pharmacy, which obtains compounds from Shanghai Haoyuan Chemexpress; [Elkabets et al., 2013]) were dissolved in DMSO. Write. We also show now that antimycin produces similar AMPK activity kinetics to both oligomycin and metformin, suggesting that this pattern of AMPK induction is common to inhibitors of mitochondrial ATP production. 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). N = 4, representative. Regulation of PFKFB3 activity by the APC/C–CDH1 proteasome system is critical for the understanding of glucose metabolism, bioenergetic supply and, possibly, the response to stress in the functioning brain. Therefore it is still possible that the sensors themselves contribute to the metabolic activities they were designed to report. The similarity of pACC patterns between parental and reporter cells, and to live-cell AMPKAR measurements, further supports the conclusion that the reporters do not substantially alter the drug-induced dynamics of AMPK activity. (H) Diagram of approximate phase relationship between NADH, AMPK, and Akt indices derived from the data collected in (F) and (G). Presumably you would be able to extract these data from previous live-cell imaging experiments, therefore allowing you to revise the paper quickly. The molecular consequences of these mutations, however, are not understood. 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. Cells expressing Peredox or AKT-KTR treated with IA also exhibited oscillations in NADH index and Akt index, respectively, with kinetics similar to those seen in the AMPK index (Figure 4D and E, Videos 5 and 6). 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. 4) A key concern is that it is unknown how relevant these fine-tuned oscillations are given the non-physiological conditions used. Next, 300 μl of ice cold ultrapure water containing 2 μg norvaline as an internal standard was added to a single well and cells were scraped into the methanol-water mixture and transferred to a microcentrifuge tube. (C) Distribution of the number of pulses in AMPK index in each cell following IA treatment, measured as the largest series of detectable pulses spaced by 1 hr or less. 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). 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. 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. Shprintzen–Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys–Dietz syndromes. To find the basis for these differences, we first examined AMPK index fluctuation over the cell cycle. This glycolytic intermediate is controlled by its own enzyme system. Imaging facilities were provided by the Nikon Imaging Center at Harvard Medical School and the Cell Biology Imaging Facility at UC Davis. One way to address this concern would be to correlate expression levels/concentrations of both the AMPK and Akt sensors with oscillation frequency. CH#04 Bioenergetics Topic# Krebs cycle (memorize just in 2 ... 29:17. Human mammary epithelial MCF10A cells clone 5E (Janes et al., 2010) were obtained from the Brugge lab and cultured as previously described (Debnath et al., 2003). 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. For example, IA-induced oscillations are most persistent at intermediate doses of 10–20 μM; at lower doses, cells successfully adapt after brief cycling, whereas at higher doses, cells simply fail to adapt and remain at a high level of AMPK activity and low NADH. An untrained adult male's leg muscle is about half of each type. 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. 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. Regulation of Glycolysis and Gluconeogenesis - Duration: 8:02. Selected ion monitoring mode was used to detect measured ions, with monitored m/z parameters identical to previously published values (Ahn and Antoniewicz, 2011). Accordingly, treatment of insulin-cultured cells with an Akt or PI3K inhibitor decreased glucose uptake and NADH index (Figure 2—figure supplement 1B–C) while increasing AMPK index (Figure 2E). However, this moderate difference between cell cycle phases does not explain the overall effect of GFs on AMPK kinetics: compared to EGF-treated cells, insulin-treated cells are more likely to be in G0/G1 but have a lower probability of AMPK index fluctuations. The GC oven was first held at 100°C for 3 min, then ramped at 2.5 °C/min to 300°C. Statistical significance was determined as described in the Materials and methods section; all inhibitor conditions differed significantly from the DMSO condition, while Torin-1 (250 nM), BMK120 (250 nM), and Rad001 (20 nM) were significantly different from BEZ235 (250 nM). Regulation of glycolysis and gluconeogenesis. We have edited the Results and Discussion section to better highlight the concept of insulin/glucose signaling as a mediator of energetic stability. AKT-KTR was constructed by fusing the N-terminal domain (amino acid residues 1–400) of human FOXO3a DNA binding mutant H212R (Tran et al., 2002; from Addgene) with a C-terminal mCherry in the retroviral pMSCV vector. (D) Fluctuation scores for each of the conditions shown in (A-C), calculated as described in the Materials and methods section. We generated MCF10A cell lines stably expressing a panel of genetically encoded fluorescent reporters for central components of the metabolic-signaling control network. The committed step is the one after which the substrate has only one way to go. […] A consultation including the Reviewer, Reviewing Editor and Senior Editor concluded that the paper could be considered again after one additional round of revision that more thoroughly addresses the question of the influence of sensor expression on metabolism. 2007). Because glycolytic intermediates feed into several other pathways, the regulation of glycolysis occurs at more than one point. 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. 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