10 Carbohydrates: Glycolysis and Glucose Homeostasis



CHAPTER


10 Carbohydrates: Glycolysis and Glucose Homeostasis












High-Yield Terms






Hexokinase/Glucokinase: glucose phosphorylating enzymes, differential tissue expression and regulatory properties, humans express 4 distinct hexokinase/glucokinase genes


PFK1: 6-phosphofructo-1-kinase, major rate-limiting enzyme of glycolysis


PFK2: bifunctional enzyme that is responsible for the synthesis of the major allosteric regulator of glycolysis via PFK1 and gluconeogenesis via fructose-1,6-bisphophatase (F-1,6-BPase)


Pyruvate kinase: multiple forms with tissue-specific distribution and regulation


PKM2: isoform of pyruvate kinase expressed in proliferating and cancer cells, participates in the Warburg effect


Substrate-level phosphorylation: refers to the formation of ATP via the release of energy from a catabolic substrate as opposed to via oxidative phosphorylation


Glucose-fatty acid cycle: describes the interrelationship between how fatty acid metabolism results in inhibition of glucose metabolism and vice versa


Intestinal glucose homeostasis: in addition to regulating glucose uptake from the diet and delivery to the blood, in times of fasting or compromised liver function, the small intestine provides up to 20% of blood glucose via gluconeogenesis using glutamine and glycerol as substrates


Renal glucose homeostasis: kidneys regulate circulating glucose levels through efficient resorption of plasma glucose as well as by being able to carry out gluconeogenesis using glutamine as a carbon source






Importance of Glycolysis


Glycolysis represents a major metabolic pathway for the conversion of the carbons of carbohydrates into other forms of biomass and for the production of cellular energy in the form of ATP. The physiologically significant property of glycolysis is that the pathway can provide cellular energy whether or not oxygen is present, as discussed later. All tissues have varying needs for the glycolytic pathway with the brain being particularly dependent upon glycolysis for energy production. Red blood cells, which lack mitochondria, are totally dependent upon glucose oxidation in glycolysis for their energy needs. In the context of glycolysis, the major carbohydrate entering the pathway is glucose. However, other carbohydrates such as fructose (see Chapter 11) and galactose (see Chapter 12) are utilized for energy and biomass production by being oxidized within the glycolytic pathway. Entry of carbohydrates into glycolysis can occur either from dietary sources, which can include a wide variety of mono-, di-, and polysaccharides, or from carbohydrate stores in the form of glycogen (see Chapter 14).


Digestion and Uptake of Dietary Carbohydrate


The details of digestive processes are discussed in Chapter 43. Digestion and absorption of carbohydrates is covered here briefly. Dietary carbohydrates enter the body in complex forms, such as mono-, di-, and polysaccharides. Through the actions of various digestive enzymes, these complex sugars are broken down into monosaccharides consisting primarily of glucose, fructose, and galactose. Intestinal absorption of carbohydrates occurs via passive diffusion, facilitated diffusion, and active transport. The primary transporter involved in the uptake of glucose is the sodium-glucose transporter 1 (SGLT1). Galactose is also absorbed from the gut via the action of SGLT1. Fructose is absorbed from the intestine via GLUT5 uptake. Indeed, GLUT5 has a much higher affinity for fructose than for glucose.


Glucose Uptake and the Role of Sugar Transporters


Glucose transporters comprise a family of at least 14 members. The most well-characterized members of the family are GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5. The glucose transporters are facilitative transporters that carry hexose sugars across the membrane without requiring energy. These transporters belong to a family of proteins called the solute carriers. Specifically, the official gene names for the GLUTs are solute carrier family 2 (facilitated glucose transporter) member. Thus, the GLUT1 gene symbol is SLC2A1, GLUT2 is SLC2A2, GLUT3 is SLC2A3, GLUT4 is SLC2A4, and GLUT5 is SLC2A5.


The glucose transporters can be divided into 3 classes based upon primary amino acid sequence comparisons:


  1. Class I transporters: GLUT1, GLUT2, GLUT3 (GLUT14 represents a duplicated GLUT3 gene), and GLUT4.


  2. Class II transporters: GLUT5, GLUT7, GLUT9, and GLUT11.


  3. Class III transporters: GLUT6, GLUT8, GLUT10, GLUT12, and HMIT (proton [H+] myoinositol symporter: SLC2A13). HMIT is also known as GLUT13.


     GLUT1 is ubiquitously distributed in various tissues with highest levels of expression seen in erythrocytes. In fact, in erythrocytes GLUT1 accounts for almost 5% of total protein. Although widely expressed, GLUT1 is not expressed in hepatocytes.


     GLUT2 is found primarily in intestine, pancreatic β-cells, kidney, and liver. The Km of GLUT2 for glucose (17 mM) is the highest of all the sugar transporters. The high Km ensures a fast equilibrium of glucose between the cytosol and the extracellular space, ensuring that liver and pancreas do not metabolize glucose until its levels rise sufficiently in the blood. GLUT2 molecules can transport both glucose and fructose. When the concentration of blood glucose increases in response to food intake, pancreatic GLUT2 molecules mediate an increase in glucose uptake, which leads to increased insulin secretion. For this reason, GLUT2 is thought to be a “glucose sensor.”


     GLUT3 is found primarily in neurons and also in the intestine. GLUT3 binds glucose with high affinity (has the lowest Km of the GLUTs), which allows neurons to have enhanced access to glucose especially under conditions of low blood glucose.


     GLUT4 predominates in insulin-sensitive tissues, such as skeletal muscle and adipose tissue. Mobilization of GLUT4 to the plasma membrane is a major function of insulin in these tissues.


     GLUT5 and the closely related transporter GLUT7 are involved in fructose transport. GLUT5 is expressed in intestine, kidney, testes, skeletal muscle, adipose tissue, and brain. Although GLUT2, -5, -7, 8, -9, -11, and -12 can all transport fructose, GLUT5 is the only transporter that exclusively transports fructose.







High-Yield Concept






Glycolysis is a major pathway for the utilization of carbohydrate carbons for redistribution into biomass as well as being oxidized for the production of ATP. The unique, and highly significant, feature of glycolysis is that it can occur in the presence (aerobic) or absence (anaerobic) of oxygen.






The Pathway of Glycolysis


The end product of glycolysis is pyruvate or lactate dependent upon the availability of oxygen. Although the reactions of glycolysis occur in the cytoplasm of all cells (Figure 10-1), the vast majority of ATP energy is generated by diversion of pyruvate into the mitochondria, where it undergoes oxidative decarboxylation and the carbons enter the TCA cycle (see Chapter 16). Under aerobic conditions, the dominant product in most tissues is pyruvate and the pathway is known as aerobic glycolysis. When oxygen is depleted, as for instance during prolonged vigorous exercise, the dominant glycolytic product in skeletal muscle is lactate and the process is known as anaerobic glycolysis. Since erythrocytes lack mitochondria, they can only carry out anaerobic glycolysis and, therefore, contribute the majority of lactate found in the blood.



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FIGURE 10-1: The pathway of glycolysis. (—PO32−; Pi, HOPO32−;, inhibition.) *Carbons 1–3 of fructose bisphosphate form dihydroxyacetone phosphate, and carbons 4–6 form glyceraldehyde 3-phosphate. The term “bis-,” as in bisphosphate, indicates that the phosphate groups are separated, whereas the term “di-,” as in adenosine diphosphate, indicates that they are joined. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.


The Individual Reactions of Glycolysis


The pathway of glycolysis can be seen as consisting of 2 separate phases. The first is the chemical priming phase requiring the input of energy in the form of ATP, and the second is considered the energy-yielding phase. In the first phase, 2 equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP). In the second phase F1,6BP is sequentially oxidized to pyruvate, with the production of 4 equivalents of ATP and 2 equivalents of NADH.


The Hexokinase Reaction


The ATP-dependent phosphorylation of glucose to form glucose 6-phosphate (G6P) is the first reaction of glycolysis, and is catalyzed by tissue-specific isoenzymes known as hexokinases. The phosphorylation accomplishes 2 goals: first, the hexokinase reaction converts nonionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars; second, the otherwise biologically inert glucose becomes activated into a labile form capable of being further metabolized.


Four mammalian isozymes of hexokinase are known (Types I-IV), with the Type IV isozyme often referred to as glucokinase. Glucokinase is the form of the enzyme found in hepatocytes and pancreatic β-cells. There are 2 major differences between hexokinases I, II, and III and glucokinase. Hexokinases have low Km for glucose (20-130 μM), are allosterically inhibited by G6P, and can utilize other hexoses (eg, fructose, mannose, glucosamine) as substrate. In contrast, glucokinase has a high Km for glucose (5-8 μM), is not feedback inhibited by G6P, and physiologically only recognizes glucose as a substrate. Glucokinase can phosphorylate other hexoses in vitro but is unable to do so at any physiologically relevant concentration of these other sugars. Although not product inhibited, hepatic glucokinase is allosterically inhibited by long-chain fatty acids (LCFA). In contrast, LCFAs do not inhibit the other forms of hexokinase. The ability of LCFAs to inhibit hepatic glucokinase is one of the mechanisms by which fatty acids inhibit glucose uptake into the liver (see discussion of Glucose-Fatty Acid Cycle later).


The kinetic properties of hepatic glucokinase allow the liver to effectively buffer blood glucose since most dietary glucose will pass through the liver and remain in the circulation for use by other tissues. After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes the liver to preferentially trap and store circulating glucose. When blood glucose falls to very low levels, since the liver is not highly dependent on glucose, it does not continue to use the meager glucose supplies that remain available.


Phosphohexose Isomerase


The second reaction of glycolysis is an isomerization, in which G6P is converted to fructose 6-phosphate, F6P. The enzyme catalyzing this reaction is phosphohexose isomerase (PHI) (also known as phosphoglucose isomerase). The reaction is freely reversible at normal cellular concentrations of the 2 hexose phosphates and thus catalyzes this interconversion during glycolysis and during gluconeogenesis.


6-Phosphofructo-1-Kinase (Phosphofructokinase-1)


The next reaction of glycolysis involves the utilization of a second ATP to convert F6P to fructose 1,6-bisphosphate (F1,6BP). This reaction is catalyzed by 6-phosphofructo-1-kinase, better known as phosphofructokinase-1 or PFK1. This reaction is not readily reversible because of its large positive free energy (ΔG0′ = +5.4 kcal/mol) in the reverse direction. The activity of PFK1 is highly regulated and as such the enzyme is considered to be the rate-limiting enzyme of glycolysis.


Aldolase A


Aldolase A (also called fructose-1,6-bisphosphate aldolase) catalyses the hydrolysis of F1,6BP into two 3-carbon products: (1) dihydroxyacetone phosphate (DHAP) and (2) glyceraldehyde 3-phosphate (G3P). The aldolase reaction proceeds readily in the reverse direction, being utilized for both glycolysis and gluconeogenesis.


Triose Phosphate Isomerase


The 2 products of the aldolase reaction equilibrate readily in a reaction catalyzed by triose phosphate isomerase (TPI1). Succeeding reactions of glycolysis utilize G3P as a substrate.


Glyceraldehyde-3-Phosphate Dehydrogenase


The second phase of glucose catabolism features the energy-yielding glycolytic reactions that produce ATP and NADH. In the first of these reactions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the NAD+-dependent oxidation of G3P to 1,3-bisphosphoglycerate (1,3BPG) and NADH. The GAPDH reaction is freely reversible.


Phosphoglycerate Kinase


The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase (PGK1). Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal physiological conditions.


Phosphoglycerate Mutase


Phosphoglycerate mutases (PGAM) represent a family of related enzymes where PGAM1 is the major glycolytic enzyme. PGAM is a heterodimer composed of a muscle (M) and/or a brain (B) isozyme, which generates MM, BB, and MB forms of the enzyme. The MM isoform predominates in muscle and the BB isoform is found at highest levels in liver, brain, and most other tissues. PGAM is responsible for converting the relatively low-energy phosphoacyl-ester of 3PG to a higher energy form, 2-phosphoglycerate, 2PG.


Enolase


Enolase (also known as phosphopyruvate hydratase) catalyzes the conversion of 2PG to phosphoenolpyruvate, PEP. PEP represents the final high-energy intermediate in glycolysis.


Pyruvate Kinase


The final reaction of aerobic glycolysis is catalyzed by the highly regulated enzyme pyruvate kinase (PK). In this strongly exergonic reaction, the high-energy phosphate of PEP is transferred to ADP-yielding ATP.


There are 2 distinct genes encoding PK activity. One encodes the liver and erythrocyte PK proteins (identified as the PKLR gene) and the other encodes the PKM proteins. The PKM gene directs the synthesis of 2 isoforms of muscle PK termed PKM1 and PKM2. The PKM gene was originally identified as the muscle pyruvate kinase gene, hence the nomenclature PKM. However, it is now known that the PKM1 protein is expressed in many tissues, whereas the PKM2 protein is most highly expressed in proliferating cells and all types of cancer.


Anaerobic Glycolysis


Under aerobic conditions, pyruvate in most cells is further metabolized via the TCA cycle. Under anaerobic conditions and in erythrocytes, which lack mitochondria, pyruvate is converted to lactate by the enzyme lactate dehydrogenase (LDH), and the lactate is transported out of the cell into the circulation. The conversion of pyruvate to lactate, under anaerobic conditions, provides the cell with a mechanism for the oxidation of NADH (produced during the GAPDH reaction) to NAD+, which occurs during the LDH-catalyzed reaction. This reduction is required since NAD+ is a necessary substrate for GAPDH, without which glycolysis will cease. Normally, during aerobic glycolysis the electrons of cytoplasmic NADH are transferred to mitochondrial carriers of the oxidative phosphorylation pathway generating a continuous pool of cytoplasmic NAD+.


The utility of anaerobic glycolysis, to a muscle cell during high physical exertion, stems from the fact that the rate of ATP production from glycolysis is approximately 100× faster than from oxidative phosphorylation. During exertion, muscle cells do not need to energize anabolic reaction pathways. The requirement is to generate the maximum amount of ATP, for muscle contraction, in the shortest time frame. This is why muscle cells derive almost all of the ATP consumed during exertion from anaerobic glycolysis.


Cytoplasmic NADH


The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative phosphorylation. Depending upon whether the malate-aspartate shuttle (Figure 10-2) or the glycerol phosphate shuttle (Figure 10-3) is used to transport the electrons from cytoplasmic NADH into the mitochondria, there will be approximately 4 or 6 moles of ATP generated for each mole of glucose oxidized to pyruvate.



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FIGURE 10-2: Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. images α-Ketoglutarate transporter and images glutamate/aspartate transporter (note the proton symport with glutamate). Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.



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FIGURE 10-3: Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.


Net Energy Yield from Glycolysis


The net yield from the oxidation of 1 mole of glucose to 2 moles of pyruvate is either 6 or 8 moles of ATP. This comprises 2 moles of ATP synthesized via substrate-level phosphorylation during the glycolytic reactions and 4 to 6 moles of ATP generated via oxidative phosphorylation from the reoxidation of cytoplasmic NADH, dependent upon which shuttle mechanism is utilized. Complete oxidation of the 2 moles of pyruvate, through the TCA cycle, yields an additional 30 moles of ATP; the total yield, therefore being either 36 or 38 moles of ATP from the complete oxidation of 1 mole of glucose to CO2 and H2O.


Lactate Metabolism


During anaerobic glycolysis the oxidation of NADH occurs through the reduction of an organic substrate. Erythrocytes and skeletal muscle (under conditions of exertion) derive all of their ATP needs through anaerobic glycolysis. The large quantity of NADH produced is oxidized by reducing pyruvate to lactate. This reaction is carried out by lactate dehydrogenase (LDH). The lactate produced during anaerobic glycolysis diffuses from the tissues and is transported to highly aerobic tissues, such as cardiac muscle and liver. The lactate is then oxidized to pyruvate in these cells by LDH and the pyruvate is further oxidized in the TCA cycle. In the liver, the pyruvate may be diverted into glucose biosynthesis via gluconeogenesis. Indeed, the connection between muscle lactate production and hepatic conversion of that lactate to glucose constitutes an important pathway referred to as the Cori cycle.


Mammalian cells contain 2 distinct types of LDH subunits, termed M and H. Combinations of these different subunits generate LDH isozymes with different characteristics. The H-type subunit predominates in aerobic tissues, such as heart muscle (as the H4 tetramer), while the M subunit predominates in anaerobic tissues, such as skeletal muscle (as the M4 tetramer). H4 LDH has a low Km for pyruvate and is also inhibited by high levels of pyruvate. The M4 LDH enzyme has a high Km for pyruvate and is not inhibited by pyruvate. This suggests that the H-type LDH is utilized for oxidizing lactate to pyruvate and the M-type the reverse.


Regulation of Glycolysis


The reactions catalyzed by hexokinase, PFK1, and PK all proceed with a relatively large free energy decrease. These nonequilibrium reactions of glycolysis would be ideal candidates for regulation of the flux through glycolysis. Indeed, all 3 enzymes are known to be allosterically controlled.


Regulation of hexokinase, however, is not the major control point in glycolysis. This is because large amounts of G6P are derived from the breakdown of glycogen (the predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and, therefore, the hexokinase reaction is not necessary. Regulation of PK is important for reversing glycolysis when ATP levels are high in order to activate gluconeogenesis. As such, this enzyme-catalyzed reaction is not a major control point in glycolysis. However, different isoforms of PK are expressed in different tissues under different conditions and as such do have an impact on glycolysis. For example, see Clinical Box 10-1 discussing the Warburg Effect and proliferating cell glycolysis. The major rate-limiting step in glycolysis is the reaction catalyzed by PFK1 (Figure 10-4).



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FIGURE 10-4: Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP). The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK1)- and fructose-1,6-bisphosphatase (F-1,6-BPase)-catalyzed reactions. PFK2 is the kinase activity and F-2,6-BPase is the phosphatase activity of the bifunctional regulatory enzyme, phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK2). PKA is cAMP-dependent protein kinase, which phosphorylates PFK2/F-2,6-BPase turning on the phosphatase activity. (+ve) and (−ve) refer to positive and negative activities, respectively. Reproduced with permission of themedicalbiochemistrypage, LLC.







CLINICAL BOX 10-1: GLYCOLYSIS IN CANCER: THE WARBURG EFFECT






In 1924, Otto Warburg made an observation that cancer cells metabolize glucose in a manner that was distinct from the glycolytic process of cells in normal tissues. Warburg discovered that, unlike most normal tissues, cancer cells tended to “ferment” glucose into lactate even in the presence of sufficient oxygen to support mitochondrial oxidative phosphorylation. This observation became known as the Warburg Effect. In the presence of oxygen, most differentiated cells primarily metabolize glucose to CO2 and H2O by oxidation of glycolytic pyruvate in the mitochondrial tricarboxylic acid (TCA) cycle. Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The ultimate fate of glucose depends not only on the proliferative state of the cell but also on the activities of the specific glycolytic enzymes that are expressed. This is particularly true for pyruvate kinase, the terminal enzyme in glycolysis (Figure 10-5). In mammals, 2 genes encode a total of 4 pyruvate kinase (PK) isoforms: the PKLR gene and the PKM gene. The PKM gene encodes the PKM1 and PKM1 isoforms. Most tissues express either the PKM1 or PKM2. PKM2 is expressed in most proliferating cells, including in all cancer cell lines and tumors. PKM2 is much less active than PKM1 but is allosterically activated by the upstream glycolytic metabolite fructose 1,6-bisphosphate (FBP). PKM2 is also unique in that it can interact with phosphotyrosine in tyrosine-phosphorylated proteins such as those resulting from growth factor stimulation of cells. The interaction of PKM2 with tyrosine-phosphorylated proteins results in the release of FBP leading to reduced activity of the enzyme. Low PKM2 activity, in conjunction with increased glucose uptake, facilitates the diversion of glucose carbons into the anabolic pathways that are derived from glycolysis. Also, in cells expressing PKM2 there is increased phosphorylation of an active site histidine (His11) in the upstream glycolytic enzyme phosphoglycerate mutase (PGAM1) which increases its mutase activity. The phosphate donor for His11 phosphorylation of PGAM1 is phosphoenolpyruvate (PEP) which is the substrate for pyruvate kinases. Phosphate transfer from PEP to PGAM1 yields pyruvate without concomitant generation of ATP. This alternate pathway allows for a high rate of glycolysis that is needed to support the anabolic metabolism observed in many proliferating cells. Targeting PKM2 for the treatment of cancers is a distinct possibility. Recent work has demonstrated that small molecule PKM2-specific activators are functional in tumor growth models in mice. These new drugs have been shown to constitutively activate PKM2 and the activated enzyme is resistant to inhibition by tyrosine-phosphorylated proteins. PKM2-specific activators reduce the incorporation of glucose into lactate and lipids. In addition, PKM2 activation results in decreased pools of nucleotide, amino acid, and lipid precursors and these effects may account for the suppression of tumorigenesis observed with these drugs.







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FIGURE 10-5: Alternative pathway of glycolysis is carried out in highly proliferative cells such as one observed in cancer cells. Cancer cells express the PKM2 isoform of pyruvate kinase, which is much less active than other isoforms and is also negatively regulated by binding to tyrosine-phosphorylated proteins. The dashed arrow for the PKM2 reaction is to demonstrate that this reaction is inefficient compared to the transfer of phosphate from PEP directly to PGAM1. PGAM1: phosphoglycerate mutase. PEP: phosphoenolpyruvate. 3-PG: 3-phosphoglycerate, 2-PG: 2-phosphoglycerate. 2,3-BPG: 2,3-bisphosphoglycerate. His11 refers to the catalytic site histidine that is phosphorylated by phosphate donation from PEP. Reproduced with permission of themedicalbiochemistrypage, LLC.


PFK1 is a tetrameric enzyme that exists in 2 conformational states termed R and T that are in equilibrium. ATP is both a substrate and an allosteric inhibitor of PFK1. Each subunit has 2 ATP-binding sites, a substrate site and an inhibitor site. The substrate site binds ATP equally well when the tetramer is in either conformation. The inhibitor site binds ATP essentially only when the enzyme is in the T state. F6P is the other substrate for PFK1 and it binds preferentially to the R state enzyme. At high concentrations of ATP, the inhibitor site becomes occupied and shifts the equilibrium of PFK1 conformation to that of the T state, thereby decreasing the ability of PFK1 to bind F6P. The inhibition of PFK1 by ATP is overcome by AMP, which binds to the R state of the enzyme and, therefore, stabilizes the conformation of the enzyme capable of binding F6P. The most important allosteric regulator of both glycolysis and gluconeogenesis is fructose 2,6-bisphosphate, F2,6BP.


Regulation of Glycolytic Flux by PFK2


The synthesis of F2,6BP is catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (simply abbreviated PFK2). The PFK2 enzyme in mammals exists as a homodimer. The PFK2 kinase domain is related to the catalytic domain of adenylate kinase. The F-2,6-BPase domain of the enzyme is structurally and functionally related to the histidine phosphatase family of enzymes. In the context of the active enzyme homodimer, the PFK2 domains function together in a head-to-head orientation, whereas the F-2,6-BPase domains can function as monomers. There are 4 PFK2 genes in mammals that each generates several isoforms. These genes are identified as PFKFB1 (liver isoform), PFKFB2 (heart isoform), PFKFB3 (brain/placenta isoform), and PFKFB4 (testes isoform).


The PFKFB1 gene expresses 3 mRNAs from 3 distinct promoters and the mRNAs and their respective promoters are called L, M, and F. The L mRNA is expressed in liver and white adipose tissue, the M mRNA is expressed in skeletal muscle and white adipose tissue, and the F mRNA is expressed in fibroblasts, proliferating cells, and fetal tissues.


The PFKFB2 gene encodes 3 mRNAs (H1, H2, and H4), all 3 of which give rise to a single-enzyme isoform. A fourth mRNA (H3) has been identified that expresses a different-sized enzyme. Although the PFKFB2 gene is referred to as the heart PFK2, none of the resultant mRNAs are strictly heart specific in their pattern of expression.


The 2 different isoforms of PFKFB3 gene are called the ubiquitous (uPFK2; also called the constitutive form) and the inducible (iPFK2) isoforms, respectively. The inducible isoform is expressed at very low levels in adult tissues but its expression is induced in tumor cell lines and by pro-inflammatory stimuli. The uPFK2 isoform has the highest kinase:bisphosphatase activity ratio.


Rapid, short-term regulation of the kinase and phosphatase activities of PFK2 is exerted by phosphorylation/dephosphorylation events. The liver isozyme is phosphorylated in an N-terminal domain by PKA. This PKA-mediated phosphorylation results in inhibition of the kinase activity while at the same time leading to activation of the phosphatase activity of PFK2. In contrast, the heart isozyme is phosphorylated at the C-terminus by several protein kinases in different signaling pathways, resulting in enhancement of the kinase activity. One of these heart kinases is AMPK and this activity allows the heart to rapidly respond to stress conditions that include ischemia. Insulin action in the heart also results in phosphorylation and activation of the kinase activity of PFK2.


Under conditions where the kinase activity of PFK2 is high, fructose flow through the PFK1-catalyzed reaction is enhanced, with a net production of F1,6BP. Conversely, when PFK2 is phosphorylated, it no longer exhibits kinase activity, but the phosphatase activity hydrolyzes F2,6BP to F6P and inorganic phosphate. The result of this change in PFK2 activity is that allosteric stimulation of PFK1 ceases, whereas in the gluconeogenic direction, allosteric inhibition of F-1,6-BPase is eliminated, and net flow of fructose through these 2 enzymes is gluconeogenic, resulting in increased glucose production.


Regulation of Glycolytic Flux by PKA


The phosphorylation of PFK2 is catalyzed by cAMP-dependent protein kinase (PKA), whose activity is, in turn, regulated by circulating peptide hormones. When blood glucose levels drop, pancreatic insulin production falls, glucagon secretion is stimulated, and circulating glucagon is highly increased. Hormones such as glucagon bind to plasma membrane receptors on liver cells, activating membrane-localized adenylate cyclase, leading to an increase in the conversion of ATP to cAMP. This newly formed cAMP binds to the regulatory subunits of PKA resulting in release and activation of the catalytic subunits. PKA phosphorylates numerous enzymes, including the PFK2. Under these conditions, the liver stops consuming glucose and becomes metabolically gluconeogenic, producing glucose to reestablish normoglycemia.


Regulation of Glycolytic Flux by Pyruvate Kinase


Regulation of glycolysis also occurs at the step catalyzed by pyruvate kinase (PK). A number of PK isozymes have been described that are derived from 2 distinct genes. Each gene can undergo alternative promoter usage or alternative splicing resulting in 4 distinct types of PK. The PKLR gene encodes the liver (PKL or L-PK) and erythrocyte (PLR or R-PK) pyruvate kinase proteins. The PKM gene encodes 2 proteins identified as PKM1 and PKM2. The designation PKM reflects the fact that the enzyme was originally thought to be muscle specific in this expression. It is now known that most tissues express either the PKM1 of the PKM2 isoform. PKM1 is found in numerous normal differentiated tissues, whereas PKM2 is expressed in most proliferating cells (see Clinical Box 10-1).


The liver isozyme (L-PK) is regulated by phosphorylation, allosteric effectors, and modulation of gene expression. L-PK is inhibited by ATP and acetyl-CoA and is activated by F1,6BP. The inhibition of L-PK by ATP is similar to the effect of ATP on PFK1. The binding of ATP to the inhibitor site reduces its affinity for PEP. The liver enzyme is also controlled at the level of synthesis. Increased carbohydrate ingestion induces the synthesis of L-PK, resulting in elevated cellular levels of the enzyme. The activity of L-PK is regulated via PKA-mediated phosphorylation, whereas the M-type isozyme found in brain, muscle, and other glucose requiring tissue is unaffected by PKA. Because of these differences, blood glucose levels and associated hormones can regulate the balance of liver gluconeogenesis and glycolysis, while muscle metabolism remains unaffected. Expression of L-PK is strongly influenced by the quantity of carbohydrate in the diet, with high-carbohydrate diets inducing up to a 10-fold increase in L-PK concentration as compared to low carbohydrate diets. L-PK is phosphorylated and inhibited by PKA, and thus it is under hormonal control similar to that described earlier for PFK2. Muscle PK (M type) is not regulated by the same mechanisms as the liver enzyme. Extracellular conditions that lead to the phosphorylation and inhibition of L-PK, such as low blood glucose and high levels of circulating glucagon, do not inhibit the muscle enzyme. The result of this differential regulation is that hormones such as glucagon and epinephrine favor liver gluconeogenesis by inhibiting liver glycolysis, while at the same time muscle glycolysis can proceed in accord with the needs directed by intracellular conditions.


In erythrocytes, the fetal PK isozyme has much greater activity than the adult isozyme; as a result, fetal erythrocytes have comparatively low concentrations of glycolytic intermediates. Because of the low steady-state concentration of fetal 1,3BPG, the 2,3BPG shunt is greatly reduced in fetal cells and little 2,3BPG is formed (Figure 10-6). Since 2,3BPG is a negative effector of hemoglobin affinity for oxygen (see Chapter 6), fetal erythrocytes have a higher oxygen affinity than maternal erythrocytes. Therefore, transfer of oxygen from maternal hemoglobin to fetal hemoglobin is favored, assuring the fetal oxygen supply. In the newborn, an erythrocyte isozyme of the M type with comparatively low PK activity displaces the fetal type, resulting in an accumulation of glycolytic intermediates. The increased 1,3BPG levels activate the 2,3BPG shunt, producing 2,3BPG needed to regulate oxygen binding to hemoglobin. Genetic diseases of adult erythrocyte PK are known in which the kinase is virtually inactive. Pyruvate kinase deficiency is the most common cause of inherited nonspherocytic hemolytic anemia. This disorder is discussed in Clinical Box 10-2.



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FIGURE 10-6: 2,3-Bisphosphoglycerate pathway in erythrocytes. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry. 29th ed. New York: McGraw-Hill; 2012.

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Jun 18, 2017 | Posted by in BIOCHEMISTRY | Comments Off on 10 Carbohydrates: Glycolysis and Glucose Homeostasis

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