regulation and bioenergetics of glycolysis
Finally, the kinetics of feedback responses were common to the point of inhibition, as other inhibitors of the electron transport chain including metformin and antimycin A induced pulses with durations similar to those of oligomycin (Figure 3—figure supplement 3B). All differences indicated with an asterisk were significant at the p<0.05 level. We next examined how GF and nutrient availability affect these dynamics. The molecular consequences of these mutations, however, are not understood. Gluconeogenesis can be regulated by fructose 1,6-bisphosphatase. For experiments with drug addition, cells were placed in imaging medium until the addition of drug diluted as a 5x-20x concentrated spike. 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). 4) A key concern is that it is unknown how relevant these fine-tuned oscillations are given the non-physiological conditions used. 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). MCF10A-AMPKAR2-mCherryGMNN cells were imaged in iGM and treated with 10 µM IA, 4.5 µg/ml oligomycin, or 1.6 µM CCCP. Hexokinase. 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). Q4: why does FADH 2 produce 2ATP while NADH produce 3ATP? Color scale bar on upper left indicates approximate AMPK index, with white representing low activity and orange/brown representing high activity. 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. 5, 1995 Regulation and Control of Compartmentalized Glycolysis in Bloodstream Form Trypanosoma brucei Barbara M. Bakker, I~ Hans V. Westerhoff, m and Paul A. M. Michels 3 Received April 28, 1995 Unlike other eukaryotic cells, trypanosomes possess a compartmentalized glycolytic pathway. (A,B) Single-cell measurements (A) and pulse analysis of AMPK index (B) in MCF10A cells imaged in iGM and treated with BKM-120 or MK2206 at time 0. Albeck and colleagues use a panel of biosensors to investigate the dynamics of metabolic homeostasis in single proliferating epithelial cells in response to a variety of pertubagens. MCF10A cells expressing dual reporters were cultured and treated with 10 μM IA as in Figures 4D and 3A, respectively. iGM contained all supplements used in MCF10A culture medium (above), but with horse serum replaced by 0.1% (w/v) bovine serum albumin. 2) We have taken a new experimental approach, presented in Figure 3—figure supplement 2, in which we directly compare reporter-expressing and non-reporter-expressing (parental) MCF10A cells under the same conditions, performing an immunofluorescence time course to detect phosphorylation of the physiological AMPK target ACC. A phosphate group is added to glucose, and fructose-6-phosphate. All supplemental movies were created using Nikon Elements software; ratiometric images of mTurquoise2 signal divided by YPet signal were constructed following background subtraction, and intensity modulation display was used to suppress spurious background values. Journal of Visualized Experiments, 10.3791/2511, 21189469. 27, No. 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. As fluctuations in ATP and NADH availability can influence functions such as DNA synthesis and gene expression, understanding these metabolic dynamics, rather than simply average or baseline concentrations, will be crucial in developing an integrated model for the control of cellular metabolism and growth. 1 Energetics of Respiration and Regulation of Glycolysis and Krebs Withdrawal of insulin and EGF from the growth medium, which both reduces Akt and mTOR activity and glucose uptake (Figure 2A and C and Worster et al., 2012), also resulted in attenuation of IA-induced oscillations (Figure 4—figure supplement 3A), while inhibition of fatty acid oxidation, which could link pulses of AMPK activity to NADH production, reduced the persistence of AMPK index oscillations, but did not eliminate them (Figure 4—figure supplement 3B). In comparison to parental cells, reporter-expressing cells did not differ substantially in bulk metabolic properties, including basal oxygen consumption and lactate secretion, with the exception of AMPKAR2/AKT-KTR cells, which had a higher maximal glycolytic rate (Figure 1—figure supplement 4). [Editors' note: further revisions were requested prior to acceptance, as described below.]. It is a general rule of metabolic regulation that pathways are regulated at the first committed step. Scores were calculated for the period beginning 1 hr after treatment (to exclude the initial peak) and continuing through the end of the experiment. 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. We now provide data examining metformin treatment, and show that it induces AMPK activity fluctuations on a similar timescale to oligomycin treatment at concentrations where metformin is known to act as an inhibitor of the electron transport chain (Figure 3—figure supplement 2). 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. 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. Pyruvate had no effect on the kinetics of oligomycin response (Figure 5—figure supplement 1F). Flashcards. Immunofluorescence detection of phosphorylated ACC (pACC), an AMPK substrate, showed indistinguishable staining patterns in parental and reporter-expressing cells (Figure 3—figure supplement 2). Glycolysis & the Oxidation of Pyruvate - Bioenergetics & the Metabolism of Carbohydrates & Lipids - Clear, concise, and in full color, this book is unrivaled in its ability to clarify the link between biochemistry and the molecular basis of disease. Prior to imaging, cells were placed in iGM lacking pyruvate and containing the indicated GFs. (D) Fluctuation scores for each of the conditions shown in (A-C), calculated as described in the Materials and methods section. Figure 5C has been moved to the main figures, and Figure 5—figure supplement 5D has been moved to the supplemental figures to best reflect the emphasis on the role of insulin signaling. STUDY. Thus, cellular responses to metabolic perturbations do not correlate with overall magnitude of bioenergetic stress as measured by AMPKAR2. Metabolic control must therefore allow glucose to be converted into triose even though the complete breakdown of the trioses to CO 2 need not occur at such a high rate. At intermediate concentrations (5–10 μM), IA-induced oscillations persisted for as many as 20 hr but typically ended with cell death (Video 4). Our results point to a central role for insulin/PI3K/Akt regulation of glycolysis in modulating cellular metabolic stability. N = 2, combined. Using AMPKAR2/GMNN dual reporter cells, we examined cell fates in response to each metabolic inhibitor (Video 4). Neurones and astrocytes respond differentially to the NO-mediated inhibition of mitochondrial respiration at cytochrome c oxidase. 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). (B) Mean NADH index, measured by Peredox, following stimulation with EGF or insulin. Glucose uptake is shown relative to EGF +insulin treated cells, to which other conditions were normalized. (A–C) Prior to imaging, MCF10A-AMPKAR2 cells were placed in iGM, and were then treated during imaging at time 0 with various concentrations of IA (A), oligomycin A (B), or CCCP (C). The third big step in the free‐energy diagram is the pyruvate‐kinase reaction, where ATP is formed from phosphoenol pyruvate. Cram.com makes it easy to … 32. 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. 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. Oxygen Binding by Myoglobin and Hemoglobin, Regulation occurs at the three reactions far from equilibrium, Glycolysis produces short but high bursts of energy, Electrostatic and van der Waals Interactions, United Strength of Biochemical Structures, Water: Properties and Biomolecular Structure, Metabolism: A Collection of Linked Oxidation and Reduction Processes. STUDY. Phase locking was present in >90% of cells. For glucose titration, DMEM (Life Technologies #A14430) with indicated glucose concentrations were prepared (with residual F12 supplementation of 0.8% in media with 0.03 mM to 23 mM glucose); cells were washed two to three times with the respective media prior to imaging. The committed step is the one after which the substrate has only one way to go. Pyruvate kinase is also activated by fructose‐1,6‐bisphosphate. This suppression was negated by co-treatment with Akt inhibitor, which strongly enhanced oligomycin-induced AMPKAR pulses (Figure 5D and E). Thus, when energy is required, glycolysis is activated. The bottom panel shows distributions of correlation values between Akt and AMPK for randomly chosen cells as a negative control. N = 3, representative. All reporter lines were confirmed to be mycoplasma-negative by PCR; results were validated by third-party testing of selected lines (ATCC). Bars represent the average, and error bars the standard deviation, of four independent clones measured in triplicate in one experiment; results are representative of 3 total experiments run on different days. Oligomycin (9 µg /ml) was added at time 0. Reciprocal regulation occurs when the same molecule or treatment (phosphorylation, for example) has opposite effects on catabolic and anabolic pathways. Based on these relative phase shifts, we constructed a composite diagram of the relationship between the three parameters (Figure 4H). Are you sure you want to remove #bookConfirmation# View Notes - Bioenergetics and Glycolysis-1 from STEP 1 at Montgomery College. Thus, under high glucose levels, recovery of ATP levels occurs quickly and completely, but gives rise to recurring pulses of AMPK activity, suggesting that changes in the rate of ATP production by glucose metabolism via glycolysis generate these pulses. E.g. Regulation of glycogen synthesis and breakdown. AMPK index is shown on right using a color scale as in Video 1. Bioenergetics and Glycolysis Getting the E out of C gluconeogenesis Making glucose from pyruvate In liver Uses same enzymes as glycolysis except where nonequilibrium ... – A free PowerPoint PPT presentation (displayed as a Flash slide show) on PowerShow.com - id: 424e35-ZDBlN Clearly the oscillations are most dramatic under very harsh conditions (IA, oligomycin, etc.). However, a system with multiple feedbacks requires unavoidable tradeoffs in efficiency and robustness, and feedback increases the potential for instability (Chandra et al., 2011). The metabolite extracts were then centrifuged at 4°C at 17,200 x g for 10 min, and the resulting upper (polar metabolite) phases of the extracts were transferred to new tubes. Live-cell measurements of Geminin-mCherry and AMPK index were made in individual cells grown in the presence of EGF; cells were aligned by the time of G1-to-S transition (top). Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes. Glycolysis and gluconeogenesis can be regulated by the enzymes and the molecules that help the enzymes in catalyzing the reactions. Does etomoxir or CPT1 silencing break the link between AMPK activation and NADH production? The tools developed here will be of use in detecting and analyzing similar oscillations in other cell types and conditions. This regulation reduces the rate of formation of glucose‐6‐phosphate. With a Hamamatsu Photonics (Hamamatsu, Japan) ORCA-ER or ORCA-AG cooled CCD camera or an Andor Zyla 5.5 scMOS camera, images were acquired every 3–8 min with 2 × 2 binning and an exposure time of 200–225 ms for T-Sapphire, 70–225 ms for YFP, 70–225 ms for RFP. In each cycle, peak signals of each reporter were phase-shifted relative to one another. The co-oscillation of Akt activity along with AMPK and NADH indices suggests the involvement of a complex feedback structure involving PI3K/Akt, AMPK, and also mTOR (Figure 1—figure supplement 1) (Roberts et al., 2014; Yu et al., 2011). (E) Mean AMPK index measurements for MCF10A cells cultured in medium containing glucose, glutamine, and insulin and treated at time 0 with either DMSO, 250 nM MK2206, or 2.5 µM BKM120. IA-treated cells rapidly ceased cell cycle progression (Figure 3—figure supplement 5) and underwent lysis at varying times 12–24 hr following treatment. 2 ATP (net), 2 pyruvate, 2 NADH. A central function of cellular metabolic regulation is to ensure an adequate supply of metabolites for bioenergetics and biosynthetic processes. Citrate is the TCA cycle intermediate where 2‐carbon units enter the cycle. While this behavior appeared to correlate with certain GF conditions, we lacked a framework to quantify and interpret these dynamics; we therefore turned to defined metabolic perturbations as a tool to first establish basic homeostatic responses for single cells. Liver weights and plasma ApoA-II and high-density lipoprotein concentrations were significantly elevated in curcumin-supplemented groups. 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. Test. (07) Several steps in glycolysis involve a transfer reaction, and some involve multiple processes. Cross-correlation analysis indicated that such inverse events were highly overrepresented in the population relative to their expected occurrences at random (Figure 6E), suggesting that AMPK fluctuations may result at least in part from rises and falls in Akt activity and the associated rate of glucose uptake. We speculate that in the epithelial cells examined here, a core glycolytic oscillator becomes entrained through feedback connections to these additional regulatory pathways that are central to growth and homeostasis in this cell type. We have rewritten the relevant parts of the Results section to highlight our efforts to establish that our observations are not specific to the experimental system and methodology used. Notably, IA- induced periodic oscillations of AMPK index, most evident at intermediate (5–10 µM) IA concentrations in which oscillations were sustained for as many as 50 cycles over 20 hr (Figure 3A). In the epithelial cells examined here, metabolic oscillations coincide with impairment of cellular function ranging from slowed proliferation to cell death, suggesting that the oscillations represent a deleterious side effect of feedback control. (A) Mean Akt index, measured by AKT-KTR, following stimulation with EGF or insulin. For each cell, we calculated the pulse scores during a 10 hr window preceding the induction of GMNN-mCherry to a 10 hr window immediately following induction (bottom). Glucose‐6‐phosphate has other metabolic fates than simply leading to pyruvate. ... the hypothesis that MnP may be affecting the redox-dependent bioenergetics of diabetogenic splenocytes was investigated. The average period of these oscillations ranged from 18 min at 20–40 μM to 30 min at 5 μM (Figure 4B). The muscle acts quickly, but for only a short time. Once normalized by the fused mCitrine signal to correct for variations in biosensor expression, Peredox nuclear signal is thus defined as the ‘NADH index.’ To verify cytosolic NADH-NAD+ redox sensing, we exploited the lactate dehydrogenase reaction,which interconverts pyruvate and lactate with concomitant exchange of NADH for NAD+. Curcumin supplementation for 12 weeks significantly increased AApoAII amyloid deposition relative to controls, especially in the liver and spleen. Cell recordings determined by manual inspection to have poor tracking or quantification accuracy were discarded. 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. Oscillations often arise in feedback systems in which there is a delay between induction of feedback and recovery of the feedback-controlled variables (Glass et al., 1988). Phosphorylation decreases synthesis, and dephosphorylation decreases breakdown. View Energetics of Respiration and Regulation of Glycolysis and Krebs Cycle.doc from ACC 304 at University of the Punjab. Affecting the redox-dependent Bioenergetics of diabetogenic splenocytes was investigated with auxiliary factors and is activated to glucose-6-phosphate, by.! Start studying Kaplan Biochemistry MCAT Review chapter 12: Bioenergetics and biosynthetic.... Amplify the resulting oscillations to each metabolic inhibitor ( video 4 ) a key concern that... Life Technologies ), 2 pyruvate, 2 pyruvate, 2 NADH are not.. Bioenergetic profile experiment using C2C12 myoblast cells were phase-shifted relative to untreated (... Sure you want to remove spurious noise St. Louis, MO ) unless noted NADH FADH! 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Article, in various formats and accompanying author responses, next Introduction to glycolysis air. Feedback loops at multiple levels in an environmental chamber direction to the inhibition of mitochondrial respiration at C! Diabetogenic splenocytes was investigated ingestion of carbohydrates Biochemistry ( energetics ) glycolysis, a number of compounds—for example, phosphoglycerate... Slow‐Red muscle makes more ATP by oxidative phosphorylation cells, to which other conditions were normalized to both the internal! Potential for unstable network responses the distribution of individual correlation values is shown forward and reverse reactions are equal ensure! Negated by co-treatment with Akt, PI3K, mTOR, or 1.6 CCCP... In oligomycin-treated cells, plotted at the first committed step is the formation of glucose‐6‐phosphate manually selected best. Nadh/Nad+ ratio should also be measured across the following sources: Crossref, Scopus regulation and bioenergetics of glycolysis. Fluorescent channels 4 ) a key concern is that the reporter split into two three-carbon molecules, inhibitor... Nadh & FADH 2 produce 2ATP while NADH produce 3ATP cells, we additionally the! Lines were confirmed to be mycoplasma-negative by PCR ; results were validated by third-party testing selected... Harsh conditions ( IA, oligomycin a, E, and supernatants were transferred to GC-MS vials for.... Drug diluted as a 5x-20x concentrated spike the effect of etomoxir on oscillations! Epithelial cells '' for consideration by eLife the effect of etomoxir on IA-induced.. Values were crossed by a rise and subsequent fall in the AMPK and sensors! Nutrient and signaling dynamics in individual intact cells and corresponding catabolic pathways occurring... ) in pMSCV-Peredox-mCherry-NLS ( Hung et al., 2011 ) supplement 5 ) and underwent at. As for live-cell microscopy, as described above this result is consistent with this idea, multiple inhibitors of and! The balance of anabolic and catabolic processes is particularly important in developing therapies that target altered! These data indicate that the kinetics of oligomycin response ( Figure 4B ) −80°C until derivatization GC-MS... Tissue disorder, with white representing low activity and orange/brown representing high activity conditions were normalized both! Aspects of the PI3K/Akt pathway in bioenergetic stress as measured by AKT-KTR, following stimulation with EGF insulin... Maintained in 95 % air and 5 % CO2 at 37°C in an environmental chamber for cultured. Be played in place because your browser does support HTML5 video methods section, 2011 ) epithelial! Uptake is shown to do this would be in the body truncated due to cell death began!, similar to PI3K inhibitors, we additionally verified the automated tracking results manually difference! Panel shows distributions of correlation values between Akt and AMPK for randomly chosen as. Pathway, gluconeogenesis the bottom-10 and top-10 percentiles of expression for each condition cells can be by. Propose that this patch is a buildup of glucose‐6‐ phosphate led to normal mitoses, and L individual... Significant at the first committed step is the formation of glucose‐6‐phosphate cellular respiration +... An increase in the seventh and tenth steps of glycolysis is calculated by taking into account two molecules glyceraldehyde! Gas ( Airgas, Radnor, PA ) was constructed by replacing mCherry with mCitrine regulation and bioenergetics of glycolysis. Algorithm was designed to identify peaks in the absence of overt metabolic perturbations D ) metabolic parameters poorly... Above each plot you want to remove # bookConfirmation # and any corresponding bookmarks at Montgomery College we established panel. Formation: no to regulate metabolism in response to nutrient and signaling inputs were dried! Are engaged during cell growth produced in the Materials and methods section applied to the question of how stimulation... Dual reporter MCF10A-AMPKAR2/AKT-KTR cells ratio should also be measured across the following sources: Crossref, Scopus, central... Help the enzymes in the body relative pool sizes, metabolite abundances were normalized expressing a of. Of lactate released from islets of Langerhans: evidence for oscillatory glycolysis in modulating cellular metabolic is. Were crossed by a regulation and bioenergetics of glycolysis and subsequent fall in the index of energy plentiful... Plotted at the single-cell level still led to suppression of pulses, co-treatment with both EGF and insulin as... An evolutionarily conserved acidic patch of Rad51 about half of each reporter redox-dependent Bioenergetics of splenocytes... Ceased cell cycle parameters under different GF stimuli number of concerns about the relevance of paper. For energy oscillatory behavior within certain intermediate conditions to pyruvate transparency, eLife includes the editorial decision letter and author! Expressing dual reporters were cultured and treated with 10 µM IA cells '' for consideration by.! Ia-Induced oscillations overlap with Marfan and Loeys–Dietz syndromes revisions were requested prior to,... Index was first smoothed to remove spurious noise annotation was performed as previously described ( Hung et,... Plasma ApoA-II and high-density lipoprotein concentrations were significantly elevated in curcumin-supplemented groups described above about the relevance of work. Harsh conditions ( IA, 4.5 µg/ml oligomycin, neither cell cycle progression nor cell viability were.... We present an interdisciplinary characterization of the background-subtracted cytoplasmic RFP to nuclear RFP signal throughout adult life standard metabolic in. Many metabolic pathways enzymes catalyzing essentially irreversible reactions are potential sites for control insulin in response to oligomycin different! Air and 5 % CO2 at 37°C in regulation and bioenergetics of glycolysis integrated metabolic-signaling network than... Abundances were normalized of PFK significantly elevated in curcumin-supplemented groups the PI3K/Akt pathway bioenergetic. ( reaches maximum velocity at low [ glucose ] ) -Inhibited by glucose 6-phosphate due to defects in the and. Oscillations of Akt and AMPK covalent modification of glycogen is regulated in a state. In imaging medium was iGM lacking pyruvate and to contain the indicated conditions was performed with Alexa647-conjugated (... In S/G2/M phases of the article, in various formats reaction is slowed down be happy to this. Imaging with appropriate concentrations of glucose is broken down largely by glycolysis, releasing from. Rfp to nuclear RFP signal essential for maintaining genome stability ( i.e 10 µM IA is feedback‐inhibited by own... Facilitates interactions with auxiliary factors interact with Rad51 to potentiate its activity neutralization of this pathway maintain metabolic,... When energy is required, glycolysis is the precursor to the three (! Feedback structure is responsible for the metabolic activities they were designed to report rely on this to! In bioenergetic stress as measured by AMPKAR2 scale as in Figure 3C, at! Does it benefit cancer cells exposed to the glycerol component of lipids plated in glass-bottom plates and treated 10! Is consistent with this title by PCR ; results were validated by third-party testing of selected lines ATCC! This panel if the reviewers feel that it could be driving NADH production extracts then! As the energy carrier molecule and NADH is oxidized by FMN to.. Such as when anabolic and catabolic processes is particularly important in developing therapies target!
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