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20.1 Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur?
Electron Transport: Electrons carried by reduced coenzymes (NADH and [FADH2] are passed through a chain of proteins and coenzymes to drive the generation of a proton gradient across the inner mitochondrial membrane Oxidative Phosphorylation: The proton gradient drives the synthesis of ATP It all happens in or at the inner mitochondrial membrane
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20.1 Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur?
Figure 20.1 (a) A drawing of a mitochondrion. (b) Tomography of a rat liver mitochondrion. The colors represent individual cristae.
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Figure 20.2 Reduction potentials for the components of the mitochondrial electrontransport chain. Values indicated are consensus values for animal mitochondria. Black bars represent o'; red bars, .
Figure 20.3 An overview of the complexes and pathways in the mitochondrial electron-transport chain.
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Figure 20.5 (a) Structural organization of mammalian Complex I, based on electron microscopy, showing functional relationships within the Lshaped complex. Electron flow from NADH to UQH2 in the membrane pool is indicated.
Figure 20.5 (b) Structure of the hydrophilic domain of Complex I from Thermus thermophilus is shown on a model of the membraneassociated complex.
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Figure 20.5 (c) Arrangement of the redox centers in Complex I. The various iron-sulfur centers of Complex I are designated by capital N.
Figure 20.5 (d) Blue arrows indicate the pathway of electron transfer from NADH to coenzyme Q. The energy of electron transfer allows a proton to cross Complex I near the interface between its hydrophilic and hydrophobic domains. Conformational changes accompanying these events push the long helical rod (magenta), reorienting residues in the associated subunits, so that protons bound there can cross into the intermembrane space.
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Figure 20.6 A scheme for electron flow in Complex II. Oxidation of succinate occurs with reduction of [FAD]. Electrons are then passed to Fe-S centers and then to CoQ.
The fatty acyl-CoA dehydrogenases are three soluble matrix enzymes involved in fatty acid oxidation (See also Chapter 23).
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Figure 20.9 The structures of iron protoporphyrin IX, heme c, and heme a.
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Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side
Cytochrome c Oxidase Electrons from cytochrome c are used in a four-electron reduction of O2 to produce 2H2O Oxygen is thus the terminal acceptor of electrons in the electron transport pathway Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2 copper sites Complex IV also transports H+ across the inner mitochondrial membrane Four H+ participate in O2 reduction and four H+ are transported in each catalytic cycle
Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side
Figure 20.13 Bovine cytochrome c oxidase consists of 13 subunits. The 3 largest subunits I (purple), II (yellow), and III (blue) contain the proton channels and the redox centers.
Subunits I, II, and III are common to most organisms. This minimal complex is sufficient to carry out both oxygen reduction and proton transport.
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Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side
Figure 20.14 The complete structure of bovine cytochrome c oxidase. As in Figure 20.13, subunit I is purple, subunit II is yellow, and subunit III is blue.
Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side Figure 20.15 The electrontransfer pathway for cytochrome c oxidase. Cytochrome c binds on the cytosolic face, transferring electrons through the copper and heme centers to reduce O2 on the matrix side of the membrane.
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Figure 20.18 (b) A model for the electron-transport pathway in the mitochondrial inner membrane. UQ/UQH2 and cyt c are mobile carriers and transfer electrons between the complexes.
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Figure 20.19 The proton and electrochemical gradients existing across the inner mitochondrial membrane.
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G = RT ln
[c2 ] + ZY [c1 ]
c1 and c2 are proton concentrations on the two sides of the membrane, Z is the proton charge, F is Faradays constant, and is the potential difference across the membrane
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Figure 20.21 (a) An axial view of the F1 unit of the ATP synthase; (b) A side view of the F1 unit with one and one subunit removed to show how the subunit (red) extends through the center of the hexamer.
In Walkers crystal structure of F1, one of the subunits contains AMP-PNP, one contains ADP, and the third site is empty the three states of Boyers model!
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Figure 20.22 ATP-ADP exchange in the absence of a proton gradient. Exchange leads to incorporation of 18O in phosphate as shown. Boyers experiments showed that 18O could be incorporated into all four positions of phosphate, demonstrating that the free energy change for ATP formation from enzymebound ADP + Pi is close to zero.
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Proton Flow Through F0 Drives Rotation of the Motor and Synthesis of ATP
Figure 20.24 (a) Protons entering the inlet halfchannel in the -subunit are transferred to binding sites on c-subunits. Rotation of the c-ring delivers protons to the outlet half-channel in the subunit. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in that synthesize ATP.
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Figure 20.27 The sites of action of several inhibitors of electron transport and oxidative phosphorylation.
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Figure 20.28 Structures of several uncouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction.
Grizzly Bear
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Some Plants Use Uncoupled Proton Transport to Raise the Temperature of Floral Spikes
Skunk Cabbage
Chipmunk
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Some Plants Use Uncoupled Proton Transport to Raise the Temperature of Floral Spikes
Philodendron
ATP-ADP Translocase Mediates the Movement of ATP & ADP Across the Mitochondrial Membrane
ATP must be transported out of the mitochondria ATP out, ADP in - through a "translocase" ATP movement out is favored because the cytosol is "+" relative to the "-" matrix But ATP out and ADP in is net movement of a negative charge out - equivalent to a H+ going in So every ATP transported out costs one H+ One ATP synthesis costs about 3 H+ Thus, making and exporting 1 ATP = 4H+
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ATP-ADP Translocase Mediates the Movement of ATP & ADP Across the Mitochondrial Membrane
ATP-ADP Translocase Mediates the Movement of ATP & ADP Across the Mitochondrial Membrane
Figure 20.29 (b) Outward transport of ATP (via the ATPADP translocase) is favored by the membrane electrochemical potential.
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20.5 - What Is the P/O Ratio for Mitochondrial Electron Transport and Oxidative Phosphorylation?
How many ATP can be made per electron pair sent through the chain? The e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen 8 H+ per turn of c8 F0 rotor 3 ATP 3.7 H+ flow back into matrix per ATP to cytosol 10/3.7 = 2.7 ATP for electrons entering as NADH For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen 6/3.7 = 1.6 ATP for electrons entering as succinate
20.6 How Are the Electrons of Cytosolic NADH Fed into Electron Transport?
Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane What to do? "Shuttle systems" effect electron movement without actually carrying NADH Glycerophosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD Malate-aspartate shuttle uses malate to carry electrons across the membrane
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Figure 20.30 The glycerophosphate shuttle couples cytosolic oxidation of NADH with mitochondrial reduction of [FAD].
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The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used
See Table 20.3 32.0 ATP per glucose if glycerol-3-P shuttle used 34.2 ATP per glucose if malate-Asp shuttle used In bacteria - no mitochondria - no extra H+ used to export ATP to cytosol. Assuming 10 c-subunits per F0 rotor (as in E. coli): 10 H+/NADH and 10 H+/3 ATP = 3 ATP/NADH 6 H+/succ and 10 H+/3ATP = ~ 1.8ATP/FADH2 (10 NADH + 2 [FADH2])/glucose 33.6 ATP
The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used
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Figure 20.32 (a) Cytochrome c is anchored at the inner mitochondrial membrane by association with cardiolipin. The peroxidase activity of cytochrome c oxidizes a cardiolipin lipid chain, releasing cytochrome c from the membrane.
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Figure 20.32 (b) The opening of pores in the outer membrane, induced by a variety of triggering agents, releases cytochrome c to the cytosol, where it initiates the events of apoptosis.
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Figure 20.33 Apaf-1 is a multidomain protein, consisting of an Nterminal CARD, a nucleotide-binding and oligomerization domain (NOD), and several WD40 domains.
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Figure 20.33 Binding of cytochrome c to the WD40 domains and ATP hydrolysis unlocks Apaf-1 to form the semi-open conformation. Nucleotide exchange leads to oligomerization and apoptosome formation.
Figure 20.33 A model of the apoptosome, a wheel-like structure with molecules of cytochrome c bound to the WD40 domains, which extend outward like spokes.
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Dr Khairul Ansari
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Gluconeogenesis is the generation (genesis) of "new (neo) glucose" from common metabolites Humans consume 160 g of glucose per day 75% of that is in the brain Body fluids contain only 20 g of glucose Glycogen stores yield 180-200 g of glucose Glycogen stores are at least partially depleted in strenuous exercise So the body must be able to make its own glucose To restore the amount stored in glycogen and to sustain normal activity
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The Substrates for Gluconeogenesis Include Pyruvate, Lactate, and Amino Acids
Pyruvate, lactate, glycerol, amino acids and all TCA intermediates can be utilized Fatty acids cannot Why? Most fatty acids yield only acetyl-CoA Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars
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Features of Gluconeogenesis
Occurs mainly in liver and kidneys Not the mere reversal of glycolysis for 2 reasons: 1) The G of glycolysis is -74 kJ/mol If gluconeogenesis were just the reverse of glycolysis, its G would be positive Energetics must change to make gluconeogenesis favorable 2) Reciprocal regulation of glycolysis and gluconeogenesis: When glycolysis is active, gluconeogenesis is turned off, and when gluconeogenesis is proceeding, glycolysis is turned off.
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The pyruvate carboxylase reaction is the initiation of the gluconeogenesis pathway Carboxylations that use bicarbonate as the carbon source require biotin as a coenzyme
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PEP carboxykinase catalyzes the conversion of oxaloacetate to PEP Lots of energy is needed to drive this reaction Energy is provided in 2 ways: Decarboxylation is a favorable reaction GTP is hydrolyzed The GTP used here is equivalent to an ATP
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The CO2 added to pyruvate in the pyruvate carboxylase reaction is removed in the PEP carboxykinase reaction. The use of GTP here (by mammals and several other organisms) is equivalent to the consumption of an ATP, due to the activity of the nucleoside diphosphate kinase (see Figure 19.2).
Fructose-1,6-bisphosphatase
Hydrolysis of F-1,6-bisPase to F-6-P Thermodynamically favorable the G in liver is -8.6 kJ/mol This is an allosterically regulated enzyme: citrate stimulates fructose-2,6-bisphosphate inhibits AMP inhibits The inhibition by AMP is enhanced by fructose-2,6bisphosphate
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Fructose-1,6-bisphosphatase
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The net reaction of conversion of pyruvate to glucose in gluconeogenesis is: 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 6 H2O glucose + 4ADP + 2 GDP + 6Pi + 2 NAD+
Net energy
G = - 37.7 KJ/mol
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Figure 22.8 The principal regulatory mechanisms in glycolysis and gluconeogenesis. Allosteric activators are indicated by plus signs and allosteric inhibitors by minus signs.
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Figure 22.11 (a) The sites of hydrolysis of starch by - and amylases are indicated.
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Figure 22.13 The glycogen phosphorylase reaction. Phosphate is the attacking nucleophile, so this reaction is a phosphorolysis.
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UDP-glucose is one of the sugar nucleotides. Discovered by Luis Leloir in the 1950s, they are activated forms of sugar.
The mechanism of the UDPglucose pyrophosphorylase reaction. Attack by a phosphate oxygen of glucose-1-P on the phosphorus of UTP is followed by departure of the pyrophosphate anion.
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Forms (1 4) glycosidic bonds in glycogen The very large glycogen particle is built around a single protein, glycogenin, at the core The first glucose is linked to a tyrosine -OH on the protein Sugar units are then added by the action of glycogen synthase Glycogen synthase transfers glucosyl units from UDP-glucose to C-4 hydroxyl at a nonreducing end of a glycogen strand. Note the oxonium ion intermediate (Fig. 22.15)
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Figure 22.16 Formation of glycogen branches by the branching enzyme. Six- or seven-residue segments of a growing glycogen chain are transferred to the C-6 hydroxyl group of a glucose residue on the same or a nearby chain.
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Figure 22.17 Insulin triggers protein kinases that stimulate glycogen synthesis.
Figure 22.17b The metabolic effects of insulin are mediated through protein phosphorylation and second messenger modulation.
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Insulin is secreted from the pancreas (to liver) in response to an increase in blood glucose Note that the portal vein is the only vein in the body that feeds an organ Insulin acts to lower blood glucose rapidly in several ways, stimulating glycogen synthesis and inhibiting glycogen breakdown
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Figure 22.18 The portal vein system carries pancreatic secretions such as insulin and glucagon to the liver.
Glucagon and Epinephrine Stimulate Glycogen Breakdown and Inhibit Glycogen Synthesis
Glucagon and epinephrine stimulate glycogen breakdown the opposite effect of insulin Glucagon (a 29-residue peptide) is also secreted by pancreas Glucagon acts in liver and adipose tissue only Epinephrine (adrenaline) is released from adrenal glands Epinephrine acts on liver and muscles When either hormone binds to its receptor on the outside surface of the cell membrane, a phosphorylase cascade amplifies the signal
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Figure 22.20 Glucagon and epinephrine activate a cascade of reactions that stimulate glycogen breakdown and inhibit glycogen synthesis in liver and muscles, respectively.
Figure 22.20 Glucagon and epinephrine each activate glycogen breakdown and inhibit glycogen synthesis in liver and muscles, respectively. In both tissues, binding of glucagon or epinephrine activates adenylyl cyclase, initiating the cascade.
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Figure 22.20. In liver, glucagon inhibits glycolysis and stimulates gluconeogenesis. In muscles, epinephrine stimulates glycolysis to provide energy for contraction.
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Figure 22.21 The effects of cortisol on carbohydrate and protein metabolism in the liver.
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Figure 22.22 The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.
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Glucose-6-P Dehydrogenase Irreversible 1st step - highly regulated! Gluconolactonase The uncatalyzed reaction happens, too 6-Phosphogluconate Dehydrogenase An oxidative decarboxylation (in that order)
Figure 22.23 The glucose-6-phosphate dehydrogenase reaction is the committed step in the pentose phosphate pathway.
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Figure 22.24 The gluconolactonase reaction. The uncatalyzed reaction also occurs.
Figure 22.25 The 6-phosphogluconate dehydrogenase reaction. Initial NADP+-dependent dehydrogenation yields a -keto acid, 3keto-6-phosphogluconate, which is very susceptible to decarboxyation (the second step). The resulting product, D-ribulose5-P, is the substrate for the nonoxidative reactions of the pentose phosphate pathway.
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Five steps, but only 4 types of reaction... Phosphopentose isomerase converts ketose to aldose Phosphopentose epimerase epimerizes at C-3 Transketolase ( a TPP-dependent reaction) transfer of two-carbon units Transaldolase (uses a Schiff base mechanism) transfers a three-carbon unit
Figure 22.26 The phosphopentose isomerase reaction converts a ketose to an aldose. The reaction involves an enediol intermediate.
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Figure 22.27 The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate. The mechanism involves an enediol intermediate and occurs with inversion at C-3.
Figure 22.28 The transketolase reaction of step 6 in the pentose phosphate pathway. This is a two-carbon transfer reaction that requires thiamine pyrophosphate as a coenzyme. TPP chemistry was discussed in Chapter 19 (see the pyruvate dehydrogenase reaction).
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Figure 22.29 The transketolase reaction of step 8 in the pentose phosphate pathway. This is another two-carbon transfer, and it also requires TPP as a coenzyme.
Figure 22.30 The mechanism of the TPP-dependent transketolase reaction. The group transferred is really an aldol. Despite this, the name transketolase persists.
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Figure 22.30 The mechanism of the TPP-dependent transketolase reaction. The group transferred is really an aldol. Despite this, the name transketolase persists.
Figure 22.31 The transaldolase reaction. In this reaction, a 3carbon unit is transferred, first to an active-site lysine, and then to the acceptor molecule.
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Figure 22.32 The transaldolase mechanism involves attack on the substrate by an active-site lysine. Departure of erythrose-4-P leaves the reactive enamine, which attacks the aldehyde carbon of Gly-3-P.
Utilization of Glucose-6-P Depends on the Cells Need for ATP, NADPH, and Rib-5-P
Glucose can be a substrate either for glycolysis or for the pentose phosphate pathway The choice depends on the relative needs of the cell for biosynthesis and for energy from metabolism ATP can be made if G-6-P is sent to glycolysis Or, if NADPH or ribose-5-P are needed for biosynthesis, G-6-P can be directed to the pentose phosphate pathway Depending on these relative needs, the reactions of glycolysis and the pentose phosphate pathway can be combined in four principal ways
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1) Both ribose-5-P and NADPH are needed by the cell In this case, the first four reactions of the pentose phosphate pathway predominate NADPH is produced and ribose-5-P is the principal product of carbon metabolism 2) More ribose-5-P than NADPH is needed by the cell Synthesis of ribose-5-P can be accomplished without making NADPH, by bypassing the oxidative reactions of the pentose phosphate pathway
Figure 22.33 When biosynthetic demands dictate, the first four reactions of the pentose phosphate pathway predominate and the principal products are ribose-5-P and NADPH.
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3) More NADPH than ribose-5-P is needed by the cell This can be accomplished if ribose-5-P produced in the pentose phosphate pathway is redirected to produce glycolytic intermediates 4) Both NADPH and ATP are needed by the cell, but ribose-5-P is not This can be done by redirecting ribose-5-P, as in case 3 above, if fructose-6-P and glyceraldehyde-3P made in this way proceed through glycolysis to produce ATP and pyruvate, and pyruvate continues through the TCA cycle to make more ATP
Integrating the Warburg Effect ATP consumption promotes cancer metabolism Cancer cells use glycolytic intermediates for the synthesis of macromolecules and must balance their ATP requirements and biosynthetic needs Xiaodong Wang has shown that EMTPD5, a UDPase in the ER is associated with ATP consumption in tumor cells UMP made in this reaction is transported into the cytosol, where it is converted back to UDP, then UTP, and then to UDP-glucose Ensuing reactions send glycolytic intermediates to the pentose phosphate pathway, helping cancer cells to fine-tune production of NADPH, ribose-5-P, and ATP
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ENTPD5 expression is enhanced in tumors and promotes ATP consumption, stimulating aerobic glycolysis. PI3K and AKT stimulate glucose uptake, as well as hexokinase and PFK. AMP kinase inhibits PFK, whereas p53 blocks synthesis of NADPH by inhibition of G6P dehydrogenase.
In addition to its role in the pentose phosphate pathway, xylulose-5-P is also a signaling molecule When blood glucose rises, glycolysis and the pentose phosphate pathways are activated in the liver The latter pathway makes xylulose-5-P, which stimulates protein phosphatase 2A (PP2A) PP2A dephosphorylates PFK-2/F-2,6-BPase and also carbohydrate-responsive element-binding protein (ChREBP) Glycolysis and lipid biosynthesis are both activated as a result
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Activation of PP2A triggers dephosphorylation of PFK-2/F2,6-BPase, which raises F-2,6-BP levels, activating glycolysis and inhibiting gluconeogenesis.
Figure 22.34
The complications of diabetes include a high propensity for cataract formation in later life Hyperglycemia is the cause, but why? Evidence points to the polyol pathway, in which glucose and other simple sugars are reduced in NADPH-dependent reactions Glucose is reduced by aldose reductase to sorbitol, which accumulates in lens fiber cells, increasing pressure and eventually rupturing the cells Aldose reductase inhibitors such as tolrestat and epalrestat suppress cataract formation
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Glucose is reduced by aldose reductase to sorbitol, which accumulates in lens fiber cells, increasing pressure and eventually rupturing the cells Aldose reductase inhibitors such as tolrestat and epalrestat suppress cataract formation
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