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One glucose molecule produces four ATP, two NADH, and two pyruvate molecules during glycolysis.
- Describe the energy obtained from one molecule of glucose going through glycolysis
- Although four ATP molecules are produced in the second half, the net gain of glycolysis is only two ATP because two ATP molecules are used in the first half of glycolysis.
- Enzymes that catalyze the reactions that produce ATP are rate-limiting steps of glycolysis and must be present in sufficient quantities for glycolysis to complete the production of four ATP, two NADH, and two pyruvate molecules for each glucose molecule that enters the pathway.
- Red blood cells require glycolysis as their sole source of ATP in order to survive, because they do not have mitochondria.
- Cancer cells and stem cells also use glycolysis as the main source of ATP (process known as aerobic glycolysis, or Warburg effect).
- pyruvate: any salt or ester of pyruvic acid; the end product of glycolysis before entering the TCA cycle
Outcomes of Glycolysis
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. For example, since the second half of glycolysis (which produces the energy molecules) slows or stops in the absence of NAD+, when NAD+ is unavailable, red blood cells will be unable to produce a sufficient amount of ATP in order to survive.
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.
Glycolysis occurs in the Cytoplasm of cells. More specifically, Glycolysis occurs in the mitochondrion, where the citric acid cycle occurs in the mitochondrial matrix, and oxidative metabolism or Glycolysis occurs at the internal folded mitochondrial membranes (cristae).
Everyone is familiar with the adage “Mitochondria is the powerhouse of the cell”, which is something everyone learns in school but doesn’t really know what it means.
The reason why Mitochondria is known as the powerhouse of the cell is because the processes of energy production happen in the Mitochondria, which include the Citric Acid cycle, or Krebs cycle and Glycolysis occur.
These two processes are the reason any cell has energy, and these processes happen across all kinds of cells in the body because all cells need energy to function as they are supposed to, grow and interact with the other cells, and this in turn causes changes in the body and keep it running.
Glycolysis can occur in aerobic (oxygenated) and anaerobic (non-oxygenated) conditions, and in both conditions it occurs in the Mitochondria.
Glycolysis is the reason behind ketosis and other types of metabolic processes that occur in cells and it is a necessary thing that people need to be aware of how they will lose weight, how much sugars or carbohydrates to consume, how to make sure they stay as healthy as possible.
There are also many medical conditions that may disrupt the occurrence of glycolysis or make use of it to cause problems, like Cancer or Diabetes.
A reason why both the krebs cycle and glycolysis occur in the cytoplasm is because one of the end products of glycolysis, Pyruvate, is one of the necessary enzymes that is fed into the Krebs cycle and its lack can lead to fumarase deficiency.
Preparatory phase of glycolysis pathway (the endothermic activation phase)
In order for glycolysis to begin, activation energy, from an ATP molecule, must be provided.
Step 1: Glucose to Glucose-6-phosphate (Hexokinase)
The first reaction of 10 glycolysis steps - substrate-level phosphorylation is catalyzed by hexokinase.
Hexokinase is one of three enzymes involved in regulation of glycolysis.
One ATP is used to phosphorylate glucose to form glucose-6-phosphate.
This step prevents the phosphorylated glucose molecule from leaving the cell because the negatively charged hydrophilic phosphate does not allow to cross the hydrophobic interior of the plasma membrane.
Trapped inside a cell in the form of glucose 6-phosphate, glucose has three metabolic options.
For example, glucose can be oxidized via glycolysis for the primary purpose of ATP production, stored as glycogen, or oxidized in the pentose phosphate pathway to generate NADPH and ribose for nucleic acid synthesis.
Three possible metabolic pathway of glucose metabolism: glycolysis, the pentose phosphate pathway, and glycogen synthesis
Step 2: Glucose-6-phosphate to Fructose-6-phosphate (Phosphohexose isomerase)
In the second step, glucose-6-phosphate molecule is then rearranged by an isomerase to form fructose-6-phosphate.
Step 3: Fructose-6-phosphate to Fructose-1,6-diphosphate (Phosphofructokinase-1)
At this point, another ATP molecule must phosphorylate the fructose-6-phosphate, producing fructose-1,6-diphosphate.
The third step of glycolysis, the phosphorylation, is catalyzed by the enzyme phosphofructokinase.
Phosphofructokinase is a rate-limiting enzyme.
- It is active when the concentration of ADP is high
- It is less active when ADP levels are low and the concentration of ATP is high.
Step 4: Fructose-1,6-diphosphate to Glyceraldehyde-3-phosphate and Dihydroxyacetone phosphate (Aldolase)
The fourth step of glycolysis utilizes an enzyme, aldolase, to split fructose-1,6-bisphosphate into one molecule of glyceraldehyde-3-phosphate (PGAL, G3P) and one molecule of dihydroxyacetone phosphate (DHAP).
Only glyceraldehyde-3-phosphate can continue through the rest of the reactions.
Step 5: Dihydroxyacetone phosphate to Glyceraldehyde-3-phosphate (Triose-phosphate isomerase)
In the fifth step, dihydroxyacetone phosphate (DHAP), which is an isomer of glyceraldehyde-3-phosphate (PGAL), is converted by an isomerase into a second molecule of PGAL.
This is the energy investment portion of the glycolysis pathway where two ATP molecules have been used.
These glyceraldehyde-3-phosphates act as the reactants for pay off phase of glycolysis.
Among different cancer treatment modalities immunotherapy enjoys the advantage of antigen-dependent specific targeting of cancer cells. Although the therapeutic potential of host immune system in affecting cancer progression has long been known (1), only in the recent decades research on the development of effective immunotherapy has gained momentum. Approval of immunotherapeutics by the Food and Drug Administration (USA) further signified immunotherapy as one of the potent and viable approaches for cancer treatment (2). During the recent expansion of the list of hallmarks of cancer, Hanahan and Weinberg (3) included “immune-evasion” and the regulation of energy metabolism” (i.e., metabolic reprogramming also referred as 𠇊ltered energy metabolism”) as additional molecular signatures of cancer. Both the regulated energy metabolism” and the immune evasion occupy similar chronological history in terms of their initial documentation (more than several decades ago) (4, 5), followed by decades of paucity and the recent recognition as cancer hallmarks (3). Emerging reports suggest that besides the historical coincidence, these two phenotypes may be functionally linked as well (6). This mini-review aims at understanding the role of tumor glycolysis in the context of immune evasion and to discuss potential immunotherapeutic implications of taming tumor glycolysis.
Metabolic flux and metabolic network analysis of Penicillium chrysogenum using 2D [13C, 1H] COSY NMR measurements and cumulative bondomer simulation
At present two alternative methods are available for analyzing the fluxes in a metabolic network: (1) combining measurements of net conversion rates with a set of metabolite balances including the cofactor balances, or (2) leaving out the cofactor balances and fitting the resulting free fluxes to measured (13)C-labeling data. In this study these two approaches are applied to the fluxes in the glycolysis and pentose phosphate pathway of Penicillium chrysogenum growing on either ammonia or nitrate as the nitrogen source, which is expected to give different pentose phosphate pathway fluxes. The presented flux analyses are based on extensive sets of 2D [(13)C, (1)H] COSY data. A new concept is applied for simulation of this type of (13)C-labeling data: cumulative bondomer modeling. The outcomes of the (13)C-labeling based flux analysis substantially differ from those of the pure metabolite balancing approach. The fluxes that are determined using (13)C-labeling data are shown to be highly dependent on the chosen metabolic network. Extending the traditional nonoxidative pentose phosphate pathway with additional transketolase and transaldolase reactions, extending the glycolysis with a fructose 6-phosphate aldolase/dihydroxyacetone kinase reaction sequence or adding a phosphoenolpyruvate carboxykinase reaction to the model considerably improves the fit of the measured and the simulated NMR data. The results obtained using the extended version of the nonoxidative pentose phosphate pathway model show that the transketolase and transaldolase reactions need not be assumed reversible to get a good fit of the (13)C-labeling data. Strict statistical testing of the outcomes of (13)C-labeling based flux analysis using realistic measurement errors is demonstrated to be of prime importance for verifying the assumed metabolic model.
Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 75-92, 2003.
S1 Fig. HMGCR inhibition promotes the proliferation of RCC cells through regulating lactate production and glucose consumption.
(A) Dose-dependent lovastatin treated on the proliferation of ACHN and 786-O cells. (B) Cell proliferation analysis in ACHN and 786-O cells treated with atorvastatin (Atr), simvastatin (Sim), or fluvastatin (Flu). (C) Western blotting analysis of HMGCR after lentiviral knockdown as exemplified in ACHN and 786-O cells. (D) Kinetic ECAR response of HMGCR knockdown or lovastatin-treated 786-O cells. (E) Glucose consumption of ACHN or 786-O cells under 24-hour lovastatin treatment. (F) Intracellular 2-NBDG glucose uptake in lovastatin-treated cells was analyzed by flow cytometry. The data are represented as the mean ± SD from 3 independent experiments, *(p ≤ 0.05), **(p ≤ 0.01), or ***(p ≤ 0.001). 2DG, 2-deoxy-D-glucose Atr, atorvastatin Ctrl, control ECAR, extracellular acidification rate Flu, fluvastatin Glu, glucose HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase Lov, lovastatin NS, non-specific Oligo, oligomycin RCC, renal cell carcinoma Sim, simvastatin wt, wild type.
S2 Fig. PKM2 is the key regulator of HMGCR-mediated glycolysis elevation.
(A) The effect of lovastatin or shHMGCR intervention on the protein levels of glycolysis-related enzymes. (B) Western blotting analysis of PKM2 after lentiviral knockdown as exemplified in ACHN cells. (C) Glucose consumption in PKM2 knockdown ACHN cells with or without lovastatin treatment. (D) Western blotting analysis of PKM2 with exogenous overexpression as exemplified in ACHN cells. (E) Glucose consumption in PKM2 overexpression ACHN cells. The data are represented as the mean ± SD from 3 independent experiments, *(p ≤ 0.05), **(p ≤ 0.01), or ***(p ≤ 0.001). Glut1, glucose transporter 1 HIF-1α, hypoxia-inducible factor-1α HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase LDHA, lactate dehydrogenase A Lov, lovastatin NS, non-specific PKM2, pyruvate kinase M2.
S3 Fig. HMGCR inhibition did not alter pkm2 expression.
pkm2 expression level in lovastatin-treated (left panel) or HMGCR knockdown (right panel) ACHN cells. The data are represented as the mean ± SD from 3 independent experiments. HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase Lov, lovastatin NS, non-specific pkm2, pyruvate kinase M2.
S4 Fig. HMGCR inhibition induces the up-regulation of HSP90.
(A) Western blotting analysis of HSP90 after lentiviral knockdown as exemplified in ACHN cells. (B) Supplemental effect of MVA pathway downstream metabolites on HSP90 level in lovastatin-treated ACHN cells. (C) Lovastatin and shHMGCR intervention increases the level of HSF-1 protein, and the effect is rescued when mevalonate is supplemented. (D) Western blotting analysis of HSP90 with exogenous overexpression as exemplified in ACHN cells. Chol, cholesterol CoQ10, coenzyme Q10 Ctrl, control FPP, farnesyl pyrophosphate GGPP, geranylgeranyl pyrophosphate HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase HSF-1, heat shock transcription factor-1 HSP90, heat shock protein 90 Lov, lovastatin MVA, mevalonate NS, non-specific.
S5 Fig. Inhibitory effect of PKM2 inhibitor in HMGCR inhibition RCC tumor.
(A) Immunohistochemical staining of CD31+ in ACHN cell xenografts (scale bar: 50 μm). (B) Extracellular lactate level, (C) glucose consumption, and (D) ECAR rate of ACHN cell with or without lovastatin and Shikonin treatment. The data are represented as the mean ± SD from 3 independent experiments, *(p ≤ 0.05), **(p ≤ 0.01) or ***p ≤ 0.001). 2DG, 2-deoxy-D-glucose ECAR, extracellular acidification rate Glu, glucose HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase Lov, lovastatin Oligo, oligomycin PKM2, pyruvate kinase M2 RCC, renal cell carcinoma SK, Shikonin wt, wild type.