Step 8. In the eighth step, the remaining phosphate group in 3-phosphoglycerate moves from the third carbon to the second carbon, producing 2-phosphoglycerate an isomer of 3-phosphoglycerate. The enzyme catalyzing this step is a mutase isomerase. Step 9. Enolase catalyzes the ninth step. This enzyme causes 2-phosphoglycerate to lose water from its structure; this is a dehydration reaction, resulting in the formation of a double bond that increases the potential energy in the remaining phosphate bond and produces phosphoenolpyruvate PEP.
Step Many enzymes in enzymatic pathways are named for the reverse reactions since the enzyme can catalyze both forward and reverse reactions these may have been described initially by the reverse reaction that takes place in vitro, under non-physiological conditions. Glycolysis starts with one molecule of glucose and ends with two pyruvate pyruvic acid molecules, a total of four ATP molecules, and two molecules of NADH.
Two ATP molecules were used in the first half of the pathway to prepare the six-carbon ring for cleavage, so the cell has a net gain of two ATP molecules and 2 NADH molecules for its use.
If the cell cannot catabolize the pyruvate molecules further via the citric acid cycle or Krebs cycle , it will harvest only two ATP molecules from one molecule of glucose. Mature mammalian red blood cells do not have mitochondria and are not capable of aerobic respiration, the process in which organisms convert energy in the presence of oxygen. Instead, glycolysis is their sole source of ATP. Therefore, if glycolysis is interrupted, the red blood cells lose their ability to maintain their sodium-potassium pumps, which require ATP to function, and eventually, they die.
Additionally, the last step in glycolysis will not occur if pyruvate kinase, the enzyme that catalyzes the formation of pyruvate, is not available in sufficient quantities. In this situation, the entire glycolysis pathway will continue to proceed, but only two ATP molecules will be made in the second half instead of the usual four ATP molecules.
Thus, pyruvate kinase is a rate-limiting enzyme for glycolysis. Privacy Policy. Skip to main content. Cellular Respiration. Search for:. Importance of Glycolysis Glycolysis is the first step in the breakdown of glucose to extract energy for cellular metabolism. Learning Objectives Explain the importance of glycolysis to cells. Table S4. Identification of proteins containing glucose regulated p-sites and concomitantly regulated at the protein level.
Table S5. The table displays a cell process enrichment analysis p-value and false discovery rate, FDR and the list of genes significantly regulated in every particular category. XLS kb. Table S6. Table S7. To identify the most differentially affected categories between groups, the standard deviation of the —Log10 p -value amongst the three groups was calculated for every GO term and then the whole ontology was sorted in decrease order of standard deviation.
The table displays a cell process enrichment analysis p -value and false discovery rate, FDR and the list of genes significantly regulated in every particular category. Figure S4. Heatmap displaying the top 30 differentially enriched ontology terms overtime considering proteins containing p-sites exclusively regulated at specific time points.
Table S8. List of genes assigned to different trajectory clusters and used for gene ontology analysis. XLSX 9 kb. Figure S5. For kinases higher KSEA positive scores in red indicates higher activity whereas negative scores in blue indicates lower activity. Conversely, for phosphatases higher KSEA positive scores in red indicates lower activity whereas negative scores in blue indicates higher activity. B Average of KSEA scores were calculated for every time point and displayed in a heatmap after hierarchical clustering.
Figure S6. KSEA output robustness. Groups of p-sites defining the regulation of specific kinases during glucose stimulation are shown in A-F. Changes p-status are plotted as changes over time.
The average changes calculated from within these groups reflect the regulation of the corresponding kinases colored lines. To confirm the signaling events revealed by KSEA, we assessed the phosphorylation status of a number of kinase substrates using phospho specific antibodies A-F. The abundance of the total protein was not affected by glucose over the time window studied here as demonstrated using antibodies recognizing the unmodified proteins.
For all phospho-specific antibodies tested, Western blotting analysis and quantitative phospho-proteomics agree regarding the changes in phosphorylation status following glucose stimulation. Figure S7. List of kinase activities according to its temporal trajectories.
Positive and negatively regulated kinases and phosphatases were clustered in three groups according with the temporal regulation. Table S9. List of drugs tested for their ability to regulate mitochondrial respiration induced by glucose. The table displays information on the drug tested, targeted mechanism, effect of the interaction and the range of concentrations tested.
Figure S8. Effect of H89 and Phenformin on basal and glucose stimulated mitochondrial respiration. Oxygen consumption recordings in INS-1E cells. All authors read and approved the final manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ornella Cominetti, Email: moc. Umberto De Marchi, Email: moc. Pedro Cutillas, Email: ku.
Andreas Wiederkehr, Email: moc. National Center for Biotechnology Information , U. Journal List Cell Commun Signal v. Cell Commun Signal. Published online Feb Author information Article notes Copyright and License information Disclaimer. Corresponding author. Received Aug 21; Accepted Feb 8. This article has been cited by other articles in PMC. Additional file 2: Figure S2. Distribution of phosphosites per protein.
PDF 4 kb. Additional file 3: Table S1. Additional file 4: Table S2. Additional file 5: Table S3. Additional file 6: Figure S3. Additional file 7: Table S4. Additional file 8: Table S5. Additional file 9: Table S6. Additional file Table S7. Additional file Figure S4. Additional file Table S8. Additional file Figure S5. Additional file Figure S6. Additional file Figure S7. Additional file Table S9. Additional file Figure S8.
Abstract Background Glucose is the main secretagogue of pancreatic beta-cells. Methods Shotgun proteomics including TiO 2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways.
Results Our kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Conclusions Our results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.
Electronic supplementary material The online version of this article Background Pancreatic beta-cells secrete the blood glucose lowering hormone insulin. Gene ontology enrichment, heatmap generation and identification of trajectory clusters Significantly regulated phosphoproteins were assigned to gene ontology GO categories for cellular processes and cellular localization using Metacore GeneGo, MI, USA.
Results Time-dependent protein phosphorylation during glucose stimulation of INS-1E cells Nutrient activation of pancreatic beta-cells occurs in several time-dependent steps. Open in a separate window. Identification of p-site clusters with similar phosphorylation kinetics Our GO cell process enrichment Fig.
Phosphorylation of proteins involved in biological processes directly linked to beta-cell activation Glucose-dependent insulin secretion engages a large number of biological processes directly involved in the full activation of beta-cells.
Dynamic regulation of kinases and phosphatases by glucose Starting from our kinetic phospho-proteomic datasets, we used KSEA [ 35 ] to infer changes in kinase activities. Glucose-regulated kinases contributing to the regulation of mitochondrial energy metabolism We hypothesized that glucose-induced signal transduction contributes to the full respiratory response beyond the role of calcium in the stimulation of beta-cell mitochondria.
Discussion Insulin secretion from the pancreatic beta-cell is biphasic. Conclusions Glucose stimulation quickly remodels the beta-cell phosphoproteome over the first hour in presence of the nutrient, affecting a large number of biological processes linked to insulin biosynthesis, early membrane trafficking, insulin granule biogenesis and transport, exocytosis and cytoskeleton dynamics.
Additional files Additional file 1: 41K, pdf Figure S1. Additional file 2: 4. Additional file 3: 29K, xlsx Table S1. Additional file 4: 38K, xlsx Table S2.
Additional file 5: 49K, xlsx Table S3. Additional file 6: K, pdf Figure S3. Additional file 7: 38K, xlsx Table S4. Additional file 8: K, xls Table S5. Additional file 9: 2. Additional file 1. Additional file 9. Additional file K, pdf Figure S5. Additional file K, pdf Figure S6. Additional file K, pdf Figure S7. Additional file K, pdf Figure S8. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files.
Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References 1. Twenty-four-hour profiles and pulsatile patterns of insulin secretion in normal and obese subjects. J Clin Invest. Rorsman P, Renstrom E. Insulin granule dynamics in pancreatic beta cells. Different pathophysiology of impaired glucose tolerance in first-degree relatives of individuals with type 2 diabetes mellitus.
EMBO J. J Physiol. Defective secretion of islet hormones in chromogranin-B deficient mice. PLoS One. Wiederkehr A, Wollheim CB. Mitochondrial signals drive insulin secretion in the pancreatic beta-cell. Mol Cell Endocrinol. Concentration dependence and time course of the effects of glucose on adenine and guanine nucleotides in mouse pancreatic islets. J Biol Chem. Mitochondrial matrix calcium is an activating signal for hormone secretion.
Cell Metab. Calcium co-regulates oxidative metabolism and ATP synthase-dependent respiration in pancreatic beta cells. To initiate glycolysis in eukaryotic cells, shown in this figure, a molecule of ATP is hydrolyzed to transfer a phosphate group to the number 6 carbon of glucose to produce glucose 6-phosphate.
The glucose 6-phosphate is rearranged to an isomeric form called fructose 6-phosphate. A second molecule of ATP is hydrolyzed to transfer a phosphate group to the number 1 carbon of fructose 6-phosphate to produce fructose 1,6-biphosphate. Step 4 The 6-carbon fructose 1,6 biphosphate is split to form two, 3-carbon molecules: glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The dihydroxyacetone phosphate is then converted into a second molecule of glyceraldehyde 3-phosphate. Step 5 As each of the two molecules of glyceraldehyde 3-phosphate are oxidized, the energy released is used to add an inorganic phosphate group to form two molecules of 1,3-biphosphoglycerate, each containing a high-energy phosphate bond.
As each of the two molecules of glyceraldehyde 3-phosphate are oxidized, the energy released is used to add an inorganic phosphate group to form two molecules of 1,3-biphosphoglycerate, each containing a high-energy phosphate bond.
As each of the two molecules of 1,3-biphosphoglycerate are converted to 3-phosphoglycerate, the high-energy phosphate group is added to ADP producing 2 ATP by substrate-level phosphorylation. The two molecules of 3-phosphoglycerate are rearranged to form two molecules of 2-phosphoglycerate. Water is removed from each of the two molecules of 2-phosphoglycerate converting the phosphate bonds to a high-energy phosphate bonds as two molecules of phosphoenolpyruvate are produced.
As the name of the enzyme suggests, this reaction involves an isomerization reaction. The reaction involves the rearrangement of the carbon-oxygen bond to transform the six-membered ring into a five-membered ring. To rearrangement takes place when the six-membered ring opens and then closes in such a way that the first carbon becomes now external to the ring. In the third step of glycolysis, fructosephosphate is converted to fructose- 1,6- bi sphosphate FBP. Similar to the reaction that occurs in step 1 of glycolysis, a second molecule of ATP provides the phosphate group that is added on to the F6P molecule.
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