Digestion of Dietary Carbohydrates
Dietary carbohydrate, from which humans gain energy, enter the body in complex forms, such as disaccharides and the polymers starch (amylose and amylopectin) and glycogen. The polymer cellulose is also consumed but not digested. The first step in the metabolism of digestible carbohydrate is the conversion of the higher polymers to the simpler, soluble monosaccharide forms that can be transported across the intestinal wall and delivered to the tissues. The breakdown of polymeric sugars begins in the mouth. Saliva has a slightly acidic pH of 6.8 and contains salivary amylase that begins the digestion of carbohydrates. The action of salivary amylase is limited to the area of the mouth and the esophagus as it is virtually inactivated by the much stronger acid pH of the stomach. Once the food has arrived in the stomach, acid hydrolysis contributes to its degradation; specific gastric and pancreatic proteases and lipases aid this process for proteins and fats, respectively. The mixture of gastric secretions, saliva, and food, known collectively as chyme, moves to the small intestine.
The main polymeric-carbohydrate digesting enzyme of the small intestine is α-amylase. This enzyme is secreted by the pancreas and has the same activity as salivary amylase, producing disaccharides and trisaccharides. The latter are converted to monosaccharides by intestinal saccharidases, including maltase that hydrolyzes di- and trisaccharides composed of glucose, and the more specific disaccharidases, sucrase-isomaltase, lactase (β-galactosidase), and trehalase. The net result is the almost complete conversion of digestible carbohydrate to its constituent monosaccharides.
The resultant glucose, fructose, and galactose are transported into the intestinal enterocytes via the actions of various carbohydrate transporters. Glucose is transported into enterocytes via the action of two transporters. One of these transporters is the Na+-dependent glucose transporter 1 (SGLT1) while the other is the Na+-independent glucose transporter 2 (GLUT2). SGLT1 is the major transporter of glucose from the lumen of the small intestine. Although GLUT2 does indeed transport glucose into intestinal enterocytes, this only occurs in response glucose-mediated translocation of intracellular vesicle-associated GLUT2, thus even in the absence of GLUT2 (such as is the case in individuals with Fanconi-Bickel disease), intestinal uptake of dietary glucose is unimpaired. 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. These monosaccharides are then transported into the circulation via the action of enterocyte GLUT2 present in the basolateral membrane. Following entry into the duodenal superior mesenteric vein the dietary sugars travel to the hepatic portal vein and then to liver parenchymal cells and other tissues of the body. Within cells, the sugars are oxidized by the various catabolic pathways of cells or they can be used as precursors for biomass production or stored as glycogen.
Oxidation of glucose is known as glycolysis. Glucose is oxidized to pyruvate or lactate. 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 many tissues is lactate and the process is known as anaerobic glycolysis. Given that erythrocytes lack mitochondria, they cannot completely oxidize glucose-derived pyruvate and instead reduce the pyruvate to lactate which enters the blood for delivery to the liver where it is used for glucose synthesis via gluconeogenesis.
The Energy Derived from Glucose Oxidation
Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the process, with the subsequent production of four equivalents of ATP and two equivalents of NADH. Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by the net production of two moles each of ATP and NADH.Glucose + 2 ADP + 2 NAD+ + 2 Pi → 2 Pyruvate + 2 ATP + 2 NADH + 2 H+
Free Energy of Reactions of Hepatic Glycolysis
Free Energy kJ/mol
Glyceraldehyde-3-phosphate dehydrogenase: GAPDH
Phosphoglycerate kinase 1
Phosphoglycerate mutase 1: PGAM1
free energy values were obtained from Li et al. (2010) “A Database of Thermodynamic Quantities for the Reactions of Glycolysis and the Tricarboxylic Acid Cycle” JPhysChemB 114(49) pp 16068-16082
The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative phosphorylation, producing either two or three equivalents (approximately) of ATP depending upon whether the glycerol phosphate shuttle or the malate-aspartate shuttle is used to transport the electrons from cytoplasmic NADH into the mitochondria. The glycerol phosphate shuttle is the principal shuttle mechanism used in adipose tissue, skeletal muscle, and brain, whereas the liver, heart, and kidneys primarily utilize the malate-aspartate shuttle.
Accurate measurements carried out in the early 1990’s demonstrated that the true yield of ATP from NADH is closer to 2.5 moles of ATP per mole of NADH and 1.5 moles of ATP per mole of FADH2. However, many texts still utilize the historical values of 3 moles of ATP per mole of NADH and 2 moles of ATP per mole of FADH2. As such the values used in this page will be the historical values.
The Malate-Aspartate Shuttle
The Gycerol Phosphate Shuttle
In order to oxidize glucose it must be activated and the activation steps, up to the generation of fructose-1,6-bisphosphate, consume two moles of ATP. Therefore, the net yield from the oxidation of one mole of glucose to two moles of pyruvate is two moles of ATP and two moles of NADH. Within the mitochondria the electrons in cytoplasmic NADH will yield 4 or 6 moles of ATP dependent upon the mitochondrial shuttle mechanism utilized.
Complete oxidation of the two moles of pyruvate, through the actions of the pyruvate dehydrogenase complex (PDHc) and the reactions of TCA cycle, yields an additional 30 moles of ATP. The total yield, therefore, of the complete oxidation of one mole of glucose to CO2 and H2O is either 36 or 38 moles of ATP. Keep in mind that these ATP yield values are obtained using the historical yield values for moles of ATP per mole of NADH and FADH2. Utilizing the newer values of ATP yield, one mole of glucose can yield either 30 or 32 moles of ATP upon complete oxidation.
The Individual Reactions of Glycolysis
The pathway of glycolysis can be seen as consisting of two separate phases. The first is the chemical priming phase requiring energy in the form of ATP, and the second is considered the energy-yielding phase. In the first phase, two equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP). In the second phase F1,6BP is degraded to pyruvate, with the production of four equivalents of ATP and two 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 isozymes known as hexokinases. The phosphorylation accomplishes two goals: First, the hexokinase reaction converts non-ionic 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 ATP-dependent hexokinase are known (Types I–IV: HK1, HK2, HK3, and HK4), with the HK4 isoform more commonly referred to as glucokinase and its gene designated as GCK. All four mammalian hexokinases/glucokinase function as monomeric enzymes. Hexokinases 1, 2, and 3 can all phosphorylate other hexose sugars (at their normal physiological concentrations) in addition to glucose, whereas glucokinase (HK4) is only physiologically active towards glucose.
The HK1 gene is located on chromosome 10q22.1 that spans 75 kb and is composed of 28 exons that generate ten alternatively spliced mRNAs that encode ubiquitously expressed and tissue-specific isoforms of the enzyme. The HK1 mRNA encodes the ubiquitously expressed isoform that is a 917 amino acid enzyme. The HK1-R isoform is the erythroid-specific isoform and is a 916 amino acid enzyme. There are four mRNAs that express the HK1-ta/tb isoform which is 921 amino acid testes-specific isoform. The HK1-td isoform is also a testes-specific isoform of 905 amino acids. The other three mRNAs encode HK1 isoform a (952 amino acids), isoform b (889 amino acids), and isoform c (885 amino acids).
The HK2 gene is located on chromosome 2p12 that spans 50 kb and is composed of 18 exons that encode a 917 amino acid protein.
The HK3 gene is located on chromosome 5q35.2 and is composed of 22 exons that encode a protein of 923 amino acids.
The GCK (HK4) gene is located on chromosome 7p13 and is composed of 13 exons. As a result of alternative promoter usage and alternative splicing the GCK gene gives rise to seven different mRNAs and seven different protein isoforms. GCK isoform 1 is a pancreas specific enzyme of 465 amino acids. GCK isoform 2 is one of two liver-specific enzymes with this protein being the predominant form found in the liver. GCK isoform 2 is a 466 amino acid protein and isoform 3 is a 464 amino acid protein. The other four GCK mRNAs (encoding isoforms 4, 5, 6, and 7) have, as yet, not been associated with tissue-specific patterns of expression.
As indicated, the HK1 isoform is ubiquitously expressed in most mammalian tissues. HK2 expression is normally restricted to insulin-sensitive tissues such as adipose tissue, skeletal and cardiac muscle. However, high level HK2 expression is observed in cancer cells and this switch is associated with poor survival rates. Activated expression of HK2 in cancer cells is associated with a loss in expression of the tumor suppressor, p53. HK3 is normally expressed at low levels. As indicated, glucokinase (HK4) expression is essentially restricted to hepatocytes and pancreatic β-cells with low expression seen within the hypothalamus. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate, i.e. only in the postprandial state.
Sigmoidal kinetics has been observed for other monomeric enzymes in cases of random addition of substrates, but this is not the case for glucokinase. The catalytic cycle for glucokinase involves an ordered addition of substrates where glucose is known to bind first followed by ATP binding. The mechanism of positive cooperativity observed for monomeric enzymes was indeed first proposed for glucokinase and is called the mnemonic model. The mnemonic mechanism of cooperativity for glucokinase involves an equilibrium between two conformational states of the enzyme that exhibit vastly different glucose affinities. Within cells there is a large predominance of the low affinity conformation of glucokinase in the absence of glucose. As glucose concentrations rise, there is a slow interconversion between the conformational states with conversion from the low affinity to the high affinity state strongly accelerated upon glucose binding to the active site of the enzyme. Only the high affinity form of glucokinase is catalytically competent, and the rate of glucose phosphorylation is very fast compared to the rate of glucose-induced conformational change. In addition to the unique kinetic parameters of glucokinase, compared to those of HK1, HK2, and HK3, glucokinase is also regulated through interaction with a regulatory protein (see the Regulation of Glycolysis section below), whereas the other three enzymes are not.
The high Km, for glucose, of hepatic glucokinase allows the liver to buffer blood glucose. After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes the liver preferentially to trap and to store circulating glucose. When blood glucose falls to very low levels, tissues such as liver and kidney, which contain glucokinases but are not highly dependent on glucose, do not continue to use the meager glucose supplies that remain available. At the same time, tissues such as the brain, which are critically dependent on glucose, continue to scavenge blood glucose using their low Km hexokinases, and as a consequence their viability is protected. Under various conditions of glucose deficiency, such as long periods between meals, the liver is stimulated to supply the blood with glucose through the pathway of gluconeogenesis. The levels of glucose produced during gluconeogenesis are insufficient to activate glucokinase, allowing the glucose to pass out of hepatocytes and into the blood.
The regulation of hexokinase and glucokinase activities is also different. Hexokinases 1, 2, and 3 are feed-back inhibited by physiological accumulation of the product (G6P) of their reactions, whereas glucokinase is not inhibited by physiological levels of G6P. The relative lack of product inhibition of glucokinase further insures liver accumulation of glucose stores during times of glucose excess, while favoring peripheral glucose utilization when glucose is required to supply energy to peripheral tissues. The activity of the hexokinases is also regulated by inorganic phosphate (Pi). HK2 and HK3 are further inhibited by Pi, whereas the G6P inhibition of HK1 is antagonized by low concentrations of Pi while high Pi concentrations contribute to further G6P inhibition of HK1.
The subcellular localization is also distinct for various hexokinase isoforms with HK1 being associated with the mitochondria while GCK moves between the nucleus and the cytosol. The HK2 enzyme can also associate with the mitochondria but it is not known if this is a physiologically relevant interaction as it is for HK1. The interaction of HK1 with the actively phosphorylating mitochondria, and its selective use of intramitochondrial ATP as a substrate is thought to facilitate coordination of glycolysis with the terminal oxidative stages of glucose metabolism which occurs within the mitochondria. This would ensure that the rate of overall glucose oxidation is commensurate with cellular energy demands while also avoiding excessive production of lactate.
The primary mechanism of glucokinase regulation is its sequestration to the nucleus by the protein, glucokinase regulatory protein, GKRP (see the Regulation of Glycolysis section below for details). Although not physiologically inhibited by its product, hepatic glucokinase is 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. The inhibition of hepatic glucose uptake by LCFA is responsible, in part, for the hyperglycemia observed in obesity.
Hypothalamic expression of the GCK gene plays an important role in the regulation of dietary glucose intake in particular, and overall feeding behavior in general. The primary hypothalamic cells expressing glucokinase are within the arcuate nucleus, ARC. Expression of the hypothalamic GCK gene increases specifically within the ARC in response to fasting. Manipulation of GCK expression within the ARC of experimental animals alters glucose intake. Increased GCK expression in the ARC results in increased glucose ingestion, whereas, decreased GCK expression results in reduced glucose ingestion. These observations indicate that ARC expression of GCK underlies the phenomenon of carbohydrate craving.
In addition to the ATP-dependent glucose phosphorylating hexokinases/glucokinase, an additional glucose phosphorylating enzyme was identified in 2004. This enzyme is dependent upon ADP for activity and not ATP. This ADP-dependent glucokinase (ADP-GK) is encoded by the ADPGK gene which is located on chromosome 15q24.1 and is composed of 9 exons that encode a 496 amino acid precursor protein. Expression of the ADPGK gene is seen in numerous tissues implying that it serves a housekeeping role with respect to glucose metabolism. The ADP-GK enzyme is highly specific for glucose with a Km for this substrate of around 0.1 mM. ADP-GK is inhibited by both high concentrations of glucose and by AMP. It is believed that this glucose phosphorylating enzyme is physiologically important during periods of ischemia/hypoxia.
he second reaction of glycolysis is an isomerization, in which G6P is converted to fructose 6-phosphate (F6P). The enzyme catalyzing this reaction is glucose-6-phosphate isomerase, GPI (also known as phosphohexose isomerase, PHI; or phosphoglucose isomerase, PGI). The reaction is freely reversible at normal cellular concentrations of the two hexose phosphates and thus catalyzes this interconversion during glycolytic carbon flow and during gluconeogenesis. The GPI gene is located on chromosome 19q13.11 spanning 40 kb and composed of 20 exons that generate seven alternatively spliced mRNAs that collectively encode five protein isoforms.
6-Phosphofructo-1-Kinase (Phosphofructokinase-1, PFK-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 PFK-1. This reaction is not readily reversible because of its large positive free energy (ΔG0′ = +5.4 kcal/mol) in the reverse direction. Nevertheless, fructose units readily flow in the reverse (gluconeogenic) direction because of the ubiquitous presence of the hydrolytic enzyme of gluconeogenesis, fructose-1,6-bisphosphatase (F-1,6-BPase).
The presence of these two enzymes in the same cell compartment provides an example of a metabolic futile cycle, which if unregulated would rapidly deplete cell energy stores. However, the activity of these two enzymes is so highly regulated that PFK-1 is considered to be the rate-limiting enzyme of glycolysis and F-1,6-BPase is considered to be the rate-limiting enzyme in gluconeogenesis.
Functional PFK-1 enzymes are tetramers composed of various combinations of three different subunits encoded by three different genes. These genes encode the muscle subunit (PFKM gene), the liver subunit (PFKL gene), and the platelet subunit (PFKP gene). The PFKM gene is located on chromosome 12q13.11 and is composed of 30 exons that generate 18 alternatively spliced mRNAs that collectively encode eight different protein isoforms. The PFKL gene is located on chromosome 21q22.3 and is composed of 28 exons that generate two alternatively spliced mRNAs. These two PFKL mRNAs encode proteins of 830 amino acids (isoform a) and 780 amino acids (isoform b). The PFKP gene is located on chromosome 10p15.2 and is composed of 28 exons that generate 11 alternatively spliced mRNAs that collectively encode eight protein isoforms.
The PFK-1 enzyme found in skeletal muscle is a homotetramer of the PFKM encoded proteins, whereas the liver PFK-1 enzyme is a homotetramer of the PFKL encoded protein. Erythrocytes contain multiple PFK-1 enzymes that randomly contain both the M and L proteins such that one can find M4 and L4 homotetramers as well as M3L1, M2L2, and M1L3 heterotetramers. In addition to platelets, fibroblasts also express the PFKP gene.
Aldolase A (Fructose-1,6-bisphosphate Aldolase):
Aldolase A catalyzes the hydrolysis of F1,6BP into two 3-carbon products: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). The aldolase A reaction proceeds readily in the reverse direction, being utilized for both glycolysis and gluconeogenesis. The aldolase A gene (gene symbol: ALDOA) is located on chromosome 16p11.2 and is composed of 17 exons that generate nine alternatively spliced mRNAs that encode two protein isoforms of 364 amino acids (isoform 1) and 418 amino acids (isoform 2).
There are three aldolase enzymes in humans, aldolase A, aldolase B, and aldolase C. The aldolase B enzyme is primarily involved in hepatic metabolism of fructose but is also expressed in the kidney and small intestine. The aldolase C enzyme is expressed primarily in the brain. The aldolase B enzyme is encoded by the ALDOB gene which is located on chromosome 9q31.1 and is composed of 9 exons that produce a protein of 364 amino acids. The aldolase C enzyme is encoded by the ALDOC gene which is located on chromosome 17q11.2 and is composed of 10 exons that produce a protein of 364 amino acids.
Triose Phosphate Isomerase:
The two products of the aldolase A reaction equilibrate readily in a reaction catalyzed by triose phosphate isomerase (TPI). Succeeding reactions of glycolysis utilize G3P as a substrate; thus, the aldolase A reaction is pulled in the glycolytic direction by mass action principals. The triose phosphate isomerase gene (gene symbol: TPI1) is located on chromosome 12p13.31 and is comprised of 8 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform.
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-P dehydrogenase (GAPDH, also abbreviated GAPD) catalyzes the NAD+-dependent oxidation of G3P to 1,3-bisphosphoglycerate (1,3BPG) with the simultaneous reduction of NAD+ to NADH. The GAPDH reaction is reversible, and the same enzyme catalyzes the reverse reaction during gluconeogenesis. Functional GAPDH enzyme exists as a homotetrameric complex. The GAPDH gene is located on chromosome 12p13.31 and is composed of 10 exons that generate five alternatively spliced mRNAs that encode three distinct protein isoforms identified as isoforms 1, 2, and 4.
The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase (PGK). Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal cell conditions. There are two PGK genes in humans identified as PGK1 and PGK2. The PGK1 gene is located on the X chromosome (Xq21.1) and is composed of 11 exons encoding a protein of 417 amino acids. The PGK2 gene arose through a retrotranspositional event with the PGK1 gene and it is expressed only in the testis. The PGK2 gene is an intronless gene located on chromosome 6p12.3 and it encodes a protein of 417 amino acids. The PGK2 encoded protein shares 87% identity to the PGK1 encoded protein.
Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3-bisphosphoglycerate, 2,3BPG (see Figure below). The pathway of red blood cell 2,3BPG synthesis is referred to as the Rapoport–Luebering shunt. The function of 2,3BPG is to serve as an important regulator of the affinity of hemoglobin for oxygen.
The synthesis of 2,3BPG, as well as its degradation to 3-phosphoglycerate, is catalyzed by the bi-functional enzyme 2,3-bisphosphoglycerate mutase (BPGM). The two activities of BPGM are 2,3-bisphosphoglycerate synthase, and 2,3-bisphosphoglycerate phosphatase. The synthase activity of the enzyme is most active at alkaline pH, whereas, the phosphatase activity is more active at acidic pH. BPGM is structurally related to the phosphoglycerate mutase (PGAM) isozymes described in the next section but is encoded by a distinct gene. The BPGM gene is located on chromosome 7q33 and is composed of 6 exons that generate three alternatively spliced mRNAs, each of which encode the same 259 amino acid protein.
The major mechanism for the control of erythrocyte 2,3BPG synthesis is the switch from normoxia to hypoxia. Under normoxic conditions approximately 75% of erythrocyte glucose is shunted to the pentose phosphate pathway for the production of NADPH in order to efficiently couple this with the anti-oxidant glutathione (GSH) cycle, thereby limiting the amount of reactive oxygen species (ROS) in the erythrocyte. However, under hypoxic condition erythrocyte glucose metabolism is switched toward the Rapoport–Luebering shunt. This shift allows for greater ATP production as well as more 2,3BPG synthesis which promotes greater levels of oxygen release to the tissues thereby countering the hypoxia..
Regulation of 2,3BPG Synthesis and High Altitude Acclimation
It has been known for more than 50 years that 2,3-BPG levels increase in the erythrocytes of normal individuals who have acclimated to life at high altitudes (hypoxic conditions). The precise molecular basis for this phenomenon has only recently been fully appreciated.
Contributing to erythrocyte metabolic reprogramming at high altitude, as well as under other hypoxic conditions, is an increase in plasma levels of adenosine. Associated with increasing adenosine concentration is increased erythrocyte synthesis of the potent bioactive lipid, sphingosine-1-phosphate (S1P). Both of these processes function in concert to reprogram metabolism in erythrocytes resulting in increased 2,3BPG synthesis..
At high altitude, as well as under hypoxic conditions, increased levels of soluble 5′-nucleotidase, ecto (encoded by the NT5E gene), a normally membrane-associated enzyme, result in increased conversion of extracellular AMP to adenosine. Adenosine exerts effects, such as in neurotransmission, by binding to specific receptors. The adenosine receptor on erythrocytes, A2B, is encoded by the ADORA2B gene. One of the responses to activation of A2B is increased PKA activity. One of the substrates of PKA is sphingosine kinase 1 (encoded by the SPHK1 gene )which leads to its activation and a resulting increase in S1P production. Combined with the actions of S1P and the activation of the A2B receptor results in increased 2,3BPG synthesis.
The most abundant membrane protein in erythrocytes was originally identified as band 3, now known as anion exchanger 1 (AE1) which is encoded by the SLC4A1 gene. The intracellular N-terminal domain of AE1 binds to several glycolytic enzymes including PFK-1, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase. One of the consequences of increased erythrocyte S1P is the release of these glycolytic enzymes from AE1 resulting in an increase in glycolysis and subsequently, increased 2,3BPG synthesis. In addition to activation of PKA, A2B activates AMPK which directly phosphorylates BPGM further enhancing synthesis of 2,3BPG.
The remaining reactions of glycolysis are aimed at converting the relatively low energy phosphoacyl-ester of 3PG to a high-energy form and harvesting the phosphate as ATP. The 3PG is first converted to 2-phosphoglycerate (2PG) by phosphoglycerate mutase (PGAM) and the 2PG conversion to phosphoenolpyruvate (PEP) is catalyzed by enolase (ENO).
There are two phosphoglycerate mutase encoding genes in humans identified as PGAM1 and PGAM2. The PGAM1 gene is ubiquitously expressed, whereas, the PGAM2 encoded protein is expressed primarily in muscle tissues and for this reason is sometimes identified as PGAM-M. The PGAM1 encoded enzyme was initially characterized from brain tissue and is, therefore, often referred to as the PGAM-B isoform. The PGAM1 gene is located on chromosome 10q24.1 and is composed of 5 exons that generate two alternatively spliced mRNAs, both of which encode distinct protein isoforms. PGAM1 isoform 1 is 254 amino acids and isoform 2 is 239 amino acids. The PGAM2 gene is located on chromosome 7p13 and is composed of 3 exons that encode a protein of 253 amino acids. Mutations in the PGAM2 gene are associated with glycogen storage disease type 10.
Biologically active PGAM enzyme is a dimeric protein that contains different proportions of subunits encoded by the PGAM1 and PGAM2 genes. The most abundant form of PGAM contains only the PGAM1 encoded protein (also identified as PGAM-BB, where the BB represents homodimeric brain isoform). The predominant form of PGAM in muscle tissues, as expected, is the PGAM-MM homodimeric isoform generated from the PGAM2 gene product.
Humans express two additional genes that are members of the PGAM gene family, neither of which encode proteins with phosphoglycerate mutase activity. The PGAM4 gene (originally identified as PGAM3) is an intronless gene located on the X chromosome (Xq21.1) within the first intron of the ATP7A gene. The PGAM4 gene appears to have arisen as a result of a retrotranspostitional event and evidence suggests that the gene encodes a potentially functional protein. The PGAM5 gene is located on chromosome 12q24.33 and encodes a protein that was originally characterized as a mitochondrially localized Ser/Thr phosphatase. More recent studies have demonstrated that the PGAM5 encoded protein also functions as a member of the histidine phosphatase family of enzymes.
There are three enolase genes in humans identified as ENO1, ENO2, and ENO3 that generate three homodimeric isoforms of functional enzyme. The proteins encoded by these three genes are referred to as α-enolase (ENO1 gene product), β-enolase (ENO3 gene product), and γ-enolase (ENO2 gene product). Expression of the ENO1 gene is ubiquitous, expression of the ENO2 gene is restricted to nervous tissues, and expression of the ENO3 gene predominates in muscle tissues.
The ENO1 gene is located on chromosome 1p36.23 and is composed of 13 exons that generate three alternatively spliced mRNAs. Two of the ENO1 generated mRNAs encode the functional 434 amino acid α-enolase protein. One of the alternatively spliced ENO1 mRNAs encodes a 341 amino acid protein identified as c-myc promoter binding protein-1 (MBP-1). The MBP-1 protein localizes to the nucleus where it functions as a tumor suppressor by acting as a transcriptional repressor of the MYC gene.
The ENO2 gene is located on chromosome 12p13.31 and is composed of 12 exons encoding a protein of 434 amino acids.
The ENO3 gene is located on chromosome 17p13.2 and is composed of 14 exons that generate three alternatively spliced mRNAs encoding two distinct proteins of 434 amino acids (isoform 1) and 391 amino acids (isoform 2). Mutations in the ENO3 gene are associated with glycogen storage disease type 13.
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 conserved as ATP. The loss of phosphate by PEP leads to the production of pyruvate in an unstable enol form, which spontaneously tautomerizes to the more stable, keto form of pyruvate. This reaction contributes a large proportion of the free energy of hydrolysis of PEP.
There are two distinct genes encoding pyruvate kinase activity. One pyruvate kinase gene, identified as the PKLR gene, is located on chromosome 1q22 and is composed of 12 exons. The PKLR gene generates two distinct mRNAs, one of which encodes the liver (PKL or L-PK) pyruvate kinase and one that encodes the erythrocyte (PLR or R-PK) pyruvate kinase. These tissue specific PK encoding mRNAs result from the use of alternative promoters. Of the 12 exons in the PKLR gene, exons 3-12 encode identical portions of both the liver and erythrocyte mRNAs. The erythrocyte PK mRNA also includes exon 1, whereas exon 2 is included in the liver PK mRNA. Activation of the promoter used in erythroid cells is driven by both basal promoter elements and a strong erythroid specific enhancer. The PKR mRNA encodes the larger of the two PK isoforms which is a 574 amino acid protein. The PKL mRNA contains an alternate 5′ exon, relative to the PKR mRNA, and the resultant encoded protein is shorter at 543 amino acids. Deficiencies in expression of the PKLR gene in erythrocytes are the cause of the most common form of inherited non-spherocytic anemia which is also the second most common cause of inherited hemolytic anemia.
The other PK gene (identified as the PKM gene) is located on chromosome 15q23 and is composed of 16 exons that generate eight alternatively spliced mRNAs. The major protein products resulting from this complex alternative splicing of the PKM precursor mRNA are identified as PKM1 (also identified as isoform b) and PKM2 (also identified as isoform a). The designation PKM reflects the fact that the enzyme was originally thought to be muscle specific in its expression. Most tissues express either the PKM1 or the PKM2 isoform. PKM1 is found in numerous normal differentiated tissues, whereas, PKM2 is expressed in most proliferating cells. All cancers that have been examined for PK expression pattern show expression of the PKM2 isoform.
The state of methylation of the PKM gene is a major mechanism for the control of expression of the PKM2 isoform. Elevated expression of the PKM2 isoform has been correlated, in numerous cancers, to a hypomethylated state in intron 1 of the PKM gene. The heightened expression of PKM2 allows for a unique pathway of altered glucose oxidation and enhanced production of lactate in cancer cells and constitute what is referred to as the Warburg effect (see below).
Under aerobic conditions, pyruvate in most cells is further metabolized via the TCA cycle. Under anaerobic conditions and in erythrocytes under aerobic conditions, 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+.
Aerobic glycolysis generates substantially more ATP per mole of glucose oxidized than does anaerobic glycolysis. The utility of anaerobic glycolysis, to a muscle cell when it needs large amounts of energy, stems from the fact that the rate of ATP production from glycolysis is approximately 100X 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.
Regulation of Glycolysis
The reactions catalyzed by hexokinase (or glucokinase), PFK-1, and PK all proceed with a relatively large free energy decrease. These non-reversible reactions of glycolysis would be ideal candidates for regulation of the flux through glycolysis. Indeed, in vitro studies have shown all three enzymes to be allosterically controlled.
Metabolomic and proteomic studies have demonstrated that in non-hepatic cells the major controls on the rate of glycolysis are exerted via the hexokinase reaction and the PFK-1 reaction. In some, but not all, cells regulation of glycolysis is also exerted through the regulation of glucose influx into the cell and lactate efflux out of the cell.
Whereas these are the critically regulated steps of glycolysis, evidence does indicate that regulation of the activity of pyruvate kinase will modify glycolytic flux. However, regulation of PK it is not a dominant contributor when compared to the other four highly regulated steps.
Regulation of Glycolysis through Glucose Influx
Increased expression of the genes encoding GLUT1 (SLC2A1 gene), GLUT3 (SLC2A3 gene), and GLUT5 (SLC2A5 gene) has been shown to lead to increased rates glycolysis. Of significance is that fact that in numerous human cancers, which have enhanced metabolic profiles and altered glycolysis, the level of expression of GLUT1 and GLUT5 is increased.
Regulation of Hepatic Glycolytic Flux by Glucokinase
A major level of control over hepatic glucokinase activity is exerted by the protein identified as glucokinase regulatory protein (GKRP) encoded by the GCKR (glucokinase regulator) gene. The GCKR gene is located on chromosome 2p23.3 and is composed of 20 exons that encode a protein of 625 amino acids. Expression of the GCKR gene is exclusively hepatic. During the fasting state, glucokinase is “held” in the nucleus by interaction with GKRP. This localization prevents glucokinase access to cytosolic glucose until it is released from GKRP. At sufficient intracellular levels of glucose, glucokinase is released from GKRP and can begin to phosphorylate cytosolic glucose.
In addition to glucose, fructose-1-phosphate (F1P), derived from the action of hepatic fructokinase phosphorylating fructose, stimulates the release of glucokinase from GKRP. Indeed, the ability of F1P to stimulate the release of glucokinase from GKRP ultimately contributes to the potentially lethal hypoglycemia associated with the fructose metabolic disorder, hereditary fructose intolerance. This latter effect results from inappropriate release of glucokinase to the cytosol leading to the phosphorylation of glucose, thereby, trapping the glucose within hepatocytes.
The activity of GKRP is also regulated by binding of fructose-6-phosphate (F6P) as well as by phosphorylation. The binding of F6P to GKRP enhances the binding of GKRP to glucokinase. This essentially prevents the generation of more glucose-6-phosphate under conditions that would be associated with rising levels of F6P such as adequate glycogen stores and adequate ATP levels within hepatocytes.
The phosphorylation of GKRP occurs through the action of AMPK which results in the release of glucokinase from GKRP. The activity of AMPK rises as the energy charge falls (increasing AMP levels), therefore, it would make sense for the hepatocyte to increase energy production via glycolysis which would be enhanced by increased glucokinase activity.
Regulation of Glycolytic Flux by PFK-1
PFK-1 is a tetrameric enzyme that exists in two conformational states termed R and T that are in equilibrium. ATP is both a substrate and an allosteric inhibitor of PFK-1. Each subunit has two 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 PFK-1 and it also binds preferentially to the R state enzyme. At high concentrations of ATP, the inhibitor site becomes occupied and shifting the equilibrium of PFK-1 conformation to that of the T state decreasing the ability of PFK-1 to bind F6P. The inhibition of PFK-1 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) which is not an intermediate in glycolysis or in gluconeogenesis.
The activity of PFK-1 is also regulated by reversible glycosylation, specifically O-GlcNAcylation. In response to hypoxia, a serine (Ser529) in PFK-1 is O-GlcNAcylated and this results in inhibition of the enzyme. This phenomenon confers a growth advantage to certain cancer cells because glucose is diverted into the pentose phosphate pathway allowing the carbon atoms to be utilized for biomass production. The pathological significance of this mode of PFK-1 regulation is that the targeting the enzyme for inhibition of the O-GlcNAcylation has been shown to inhibit cancer growth and impairs tumor formation.
Regulation of Glycolytic Flux by PFK-2
The synthesis of F2,6BP is catalyzed by the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/F-2,6-BPase, or commonly just PFK-2). PFK-2 in mammalian organisms is a homodimer. The PFK-2 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 PFK-2 domains function together in a head-to-head orientation, whereas the F-2,6-BPase domains can function as monomers. The PFK-2 reaction is catalyzed in the N-terminal half of the enzyme subunit, whereas the FBPase-2 reaction is catalyzed in the C-terminal half. There are four PFK-2 isozymes in mammals, each coded for by a different gene. Each PFK-2 gene also encodes several different isoforms via alternative splicing. The four different PFK-2 isoforms are expressed in the liver, heart, brain (or placenta) and testis and each differs by the sequences of their bifunctional catalytic cores and their N-terminal amino acid sequences.
Each of the different PFK-2 genes has been characterized. The PFKFB1 gene, located on the X chromosome (Xp11.21) and composed of 17 exons, encodes the liver isozyme. The PFKFB2 gene is found on chromosome 1q32.1 and is composed of 16 exons that encode the heart isozyme. The PFKFB3 gene is located on chromosome 10p15.1 and is composed of 19 exons that encode the brain/placenta isoform. The PFKFB4 gene is located on chromosome 3p21.31 and is composed of 19 exons that encode the testis isozyme. High level expression of the PFKFB4 gene is also seen in cancer cells and in response to hypoxia. The regulatory sequences present in these four genes have been identified that are responsible for their long-term control by hormones and tissue specific transcription factors. The PFKFB1 and PFKFB2 genes are the most highly characterized of the four.
The PFKFB1 gene is composed of 17 exons spanning 60 kbp and encodes three different mRNAs as a result of alternative splicing and alternative promoter usage. These mRNAs, and their promoters, are called L, M and F. The three mRNAs differ in their 5′ ends but share 12 common exons (exons 2-13), six of which encode the PFK-2 catalytic domain and six of which encode the F-2,6-BPase catalytic domain. The L-type exon 1 is found the the L mRNA and the M-type exon 1 is found in the M mRNA. There are two F-type mRNAs that are derived by the splicing of two non-coding exons to part of the M-type exon 1. The L-type exon 1 sequences included in the L type enzyme contain a serine residue (Ser32) that is the target of PKA-mediated phosphorylation (see below). 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 is composed of 20 exons spanning 22 kbp that encode at least four mRNAs as a result of alternative promoter usage. Exons 3-14 are very similar to those of the PFKFB1 gene that code for the core catalytic domain. Exon 15 contains several phosphorylation sites. How the distinct 5′ ends relate to the three mRNAs (H1, H2 and H4) that give rise to the 58 kDa isoform and the mRNA (H3) that encodes the 54 kDa isoform, is as yet unknown. Additionally, none of these mRNAs are strictly heart-specific in their pattern of expression.
The PFKFB3 gene is composed of 16 exons. Alternative splicing of exon 15 and possibly differential promoter usage yields two main isoforms that differ by a short C-terminal sequence. These two different PFKFB3 isoforms are referred to as the ubiquitous isoform (uPFK-2; also called the constitutive form) and the inducible isoform (iPFK-2). 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 uPFK-2 isoform has the highest kinase/bisphosphatase activity ratio.
Of potential clinical significance is the fact that studies of iPFK-2 function indicate that the adipose tissue enzyme may play a role in the concept of healthy obesity. The vast majority of obese individuals will develop type 2 diabetes (T2D) and various cardiovascular diseases such as atherosclerosis. However, it has always been a scientific and clinical curiosity that a small percentage of overweight or obese individuals do not develop these same symptoms, the so-called healthy obese. In addition, it is known that certain thinner individuals may develop the types of health problems more typical of those associated with obesity. When iPFK-2 expression is knocked-out in mice there is a reduction in diet-induced obesity but the negative consequences include an exacerbation of adipose tissue inflammation and enhanced insulin resistance. This observation led researchers to speculate that iPFK-2 expression may link metabolic and inflammatory responses and, therefore, could underlie the healthy obesity concept. Results from the converse experiment does indeed strengthen the idea of iPFK-2 underlying healthy obesity. When iPFK-2 is overexpressed in adipose tissue there results an increase in fat deposition in adipose tissue which equates with obesity. However, these mice have suppressed inflammatory responses along with improved insulin sensitivity in both adipose tissue and the liver. The latter being equated with healthy obesity.
Rapid, short-term regulation of the kinase and phosphatase activities of PFK-2 are exerted by phosphorylation and dephosphorylation events. The liver isoform is phosphorylated at the N-terminus on Ser32, adjacent to the PFK-2 domain, by PKA. This PKA-mediated phosphorylation results in inhibition of the PFK-2 activity while at the same time leading to activation of the F-2,6-BPase activity. In contrast, the heart isoform is phosphorylated at the C-terminus by several protein kinases in different signaling pathways, resulting in enhancement of the PFK-2 activity. One of these heart kinases is AMPK and this activity allows the heart to respond rapidly to stress conditions that include ischemia. Insulin action in the heart also results in phosphorylation and activation of the PFK-2 activity of the enzyme. This insulin-mediated effect is, in part, the result of the activation of PDK1 (PIP3-dependent protein kinase). For more information on the signaling pathways initiated by the actions of insulin go to the Insulin Functions page.
Under conditions where PFK-2 is active, fructose flow through the PFK-1/F-1,6-BPase reactions takes place in the glycolytic direction, with a net production of F1,6BP. When the bifunctional enzyme is phosphorylated it no longer exhibits kinase activity, but a new active site hydrolyzes F2,6BP to F6P and inorganic phosphate. The metabolic result of the phosphorylation of the bifunctional enzyme is that allosteric stimulation of PFK-1 ceases, allosteric inhibition of F-1,6-BPase is eliminated, and net flow of fructose through these two enzymes is gluconeogenic, producing F6P and eventually glucose.
Regulation of Glycolytic Flux by PKA
The interconversion of the bifunctional enzyme is catalyzed by cAMP-dependent protein kinase (PKA), which in turn is 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 (see diagram below). cAMP binds to the regulatory subunits of PKA, leading to release and activation of the catalytic subunits. PKA phosphorylates numerous enzymes, including the bifunctional PFK-2/F-2,6-BPase. 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). There are four distinct isoforms of PK in human tissues encoded for by two different genes. One is located on chromosome 1, identified as the PKLR gene, and it encodes the liver (PKL or L-PK) and erythrocyte (PKR or R-PK) pyruvate kinase proteins. Expression of PKL or PKR is dependent upon the use of tissue-specific promoter elements in the PKLR gene. The other pyruvate kinase gene is located on chromosome 15 and encodes two proteins identified as PKM1 and PKM2. The designation PKM reflects that fact that the enzyme was originally thought to be muscle specific in is expression.
The two PKM isoforms result from alternative splicing of the PKM gene. 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. All cancers that have been examined for PK expression pattern show expression of the PKM2 isoform. Indeed, expression of PKM2 allows for a unique pathway of enhanced glucose oxidation to lactate in cancer cells.
The liver isoform (PKL or L-PK) has been most studied in vitro. This enzyme is inhibited by ATP and acetyl-CoA and is activated by F1,6BP. The inhibition of PK by ATP is similar to the effect of ATP on PFK-1. 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. Regulation of L-PK is characteristic of a gluconeogenic tissue being regulated via phosphorylation by PKA. Whereas the M-type isozymes are unaffected by PKA. As a consequence of these differences, blood glucose levels and associated hormones can regulate the balance of liver gluconeogenesis and glycolysis while for instance, muscle metabolism remains unaffected.
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 (see diagram above) is greatly reduced in fetal cells and little 2,3BPG is formed. Since 2,3BPG is a negative effector of hemoglobin affinity for oxygen, 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. The erythrocytes of affected individuals have a greatly reduced capacity to make ATP and thus do not have sufficient ATP to perform activities such as ion pumping and maintaining osmotic balance. These erythrocytes have a short half-life due to easy lysis. Pyruvate kinase deficiency is the most common cause of inherited non-spherocytic hemolytic anemia and the second most common cause of inherited hemolytic anemia behind glucose-6-phosphate dehydrogenase (G6PD) deficiencies.
The liver PK isozyme is regulated by phosphorylation, allosteric effectors, and modulation of gene expression. The major allosteric effectors are F1,6BP, which stimulates PK activity by decreasing its Km for PEP, and for the negative effector, ATP. Expression of the liver PK gene is strongly influenced by the quantity of carbohydrate in the diet, with high-carbohydrate diets inducing up to 10-fold increases in PK concentration as compared to low carbohydrate diets. Liver PK is phosphorylated and inhibited by PKA, and thus it is under hormonal control similar to that described earlier for PFK-2.
Muscle PK (PKM1) is not regulated by the same mechanisms as the liver enzyme. Extracellular conditions that lead to the phosphorylation and inhibition of liver 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 needs directed by intracellular conditions.
Regulation of Glycolysis through Lactate Efflux
Lactate is transported out of cells, such as erythrocytes and skeletal muscle, via one of several members of the monocarboxylate transporter (MCT) family. These lactate transporters include MCT1 (SLC16A1 gene), MCT2 (SLC16A7 gene), MCT3 (SLC16A8 gene), MCT4 (SLC16A3 gene), MCT5 (SLC16A4 gene), MCT6 (SLC16A5 gene), MCT7 (SLC16A6 gene), and MCT9 (SLC16A9 gene). Increased expression of the gene (SLC16A3) encoding MCT4 has been shown to be associated with increased flux of glucose through glycolysis in numerous human cancers.
Regulation of Glycolysis by Ubiquitylation
Experiments in cancer cells, which have elevated rates of glycolysis, have demonstrated that the expression of several genes encoding deubiquitylating enzymes (DUB) is upregulated in concert with most of the genes encoding glycolytic enzymes. The DUB that were identified include JOSD2 (josephin domain containing 2), USP5 (ubiquitin specific peptidase 5), STAMBP (signal transducing adapter molecule binding protein), PSMD7 (proteasome 26S subunit non-ATPase 7), and PSMD14. The significance of JOSD2 is that it is a positive regulator of glycolysis. Indeed, knockdown of JOSD2 levels in cancer cells reduces the rate of glycolysis. This effect is coincident with reduced abundance of the glycolytic enzymes, aldolase A, PFK-1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate isomerase (GPI), lactate dehydrogenase (both LDHA and LDHB), and the serine metabolic enzyme, phosphoglycerate dehydrogenase (PHGDH.
Evidence has demonstrated that the glycolytic enzymes, aldolase A and PFK-1, and the serine synthesis enzyme, phosphoglycerate dehydrogenase, exist in a complex. The JOSD2-mediated deubiquitylation of aldolase A, and in particular PFK-1, directly results in increased flux of glucose into the glycolytic pathway. With respect to cancer cell metabolism this is significant given that targeting the activity of JOSD2 could be therapeutic since its loss would lead to significantly reduced rates of glycolysis in these cells.
Glucose Metabolism and Cancer: The Warburg Effect
It has been known for over 75 years that cancer cells metabolize glucose differently than differentiated cells. In 1924, Otto Warburg made an observation that cancer cells undertook glucose metabolism 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 TCA cycle. Originally postulated to be the result of mitochondrial dysfunction in cancer cells, it was subsequently shown that this was not the mitigating reason for the increased production of lactate in cancer cells. It might seem counterintuitive that highly proliferative cells, such as is characteristic of cancers, would bypass oxidation of pyruvate (from glucose) in mitochondria since this is the organelle where the vast majority of the ATP from glycolysis is generated. However, it has been shown that oncogenic mutations can result in the uptake of nutrients, particularly glucose and glutamine, that meet or exceed the bioenergetic demands of cell growth and proliferation. 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.
Pyruvate Kinase Isoforms and Cancer
In mammals, two genes encode a total of four pyruvate kinase (PK) isoforms. The PKLR gene encodes the liver (L-PK or PKL) and the erythrocyte (R-PK or PKR) isoforms of pyruvate kinase via a process of alternative promoter usage. The PKM gene, so called originally due to initial characterization in muscle tissues, encodes the PKM1 and PKM2 isoforms. PKM1 and PKM2 are derived via alternative splicing of the PKM gene encoded mRNA. This results in mutual exclusion of a single conserved exon encoding 56 amino acids. In PKM1 mRNA exon 9 is included, whereas it is excluded from PKM2 mRNA. In the PKM2 mRNA exon 10 is included, whereas it is excluded from PKM1 mRNA. Most tissues express either the PKM1 or PKM2. PKM1 is found in many normal differentiated tissues, whereas PKM2 is expressed in most proliferating cells, including in all cancer cell lines and tumors tested to date.
Although PKM1 and PKM2 are highly similar in amino acid sequence they have different catalytic and regulatory properties. PKM1 has high constitutive enzymatic activity and is exclusively an enzyme that utilizes the energy of phosphoenolpyruvate (PEP) to phosphorylate ADP to ATP while converting PEP to pyruvate as is typical of glycolysis.
PKM2 Structure-Function Relationships
In contrast to PKM1, PKM2 activities are more complex and, in part, depend on the oligomeric state of the enzyme. PKM2 can exist as a homodimeric or as a homotetrameric enzyme. The tetrameric form of PKM2 exhibits high catalytic activity in the conversion of phosphoenolpyruvate (PEP) to pyruvate and this is associated with ATP synthesis and catabolic metabolism of glucose. In contrast, the dimeric form of PKM2 has low catalytic activity and this low activity, in part, facilitates the diversion of glycolytic intermediates into glucose metabolic branch pathways that including glycerol synthesis and the pentose phosphate pathway. Of significance to proliferating cells as well as cancer is that the NADPH produced by the pentose phosphate pathway is used to suppress reactive oxygen species (ROS) production and the ribose-5-phosphate is used for nucleotide synthesis.
The regulation of the dimeric versus tetrameric forms of PKM2 is under complex control. The upstream glycolytic metabolite fructose 1,6-bisphosphate (FBP) was the first characterized allosteric regulator of PKM2 which promotes the formation of the tetrameric form of the enzyme. The amino acid serine, which can be derived from the glycolytic intermediate, 3-phosphoglycerate is also an allosteric activator promoting the tetrameric form of PKM2. Another allosteric stimulator of the tetrameric PKM2 is 5-aminoimidazole-4-carboxyribonucleotide (SAICAR) which is an intermediate in purine nucleotide biosynthesis.
Post-translational Modification of PKM2
Post-translational modifications that include phosphorylation and acetylation promote the less active dimeric form of PKM2. In growth factor stimulated proliferative cells and in cancer cells there is increased tyrosine phosphorylation of numerous proteins including PKM2. When PKM2 is phosphorylated on tyrosine (Y105) it releases FBP causing conversion to the less active dimeric form. Low PKM2 activity, in conjunction with increased glucose uptake, facilitates the diversion of glucose carbons into the anabolic pathways that are derived from glycolysis. PKM2 is also inhibited by direct oxidation of a cysteine residue (Cys358) as an adaptive response to increased intracellular reactive oxygen species (ROS). This inhibition does not occur in PKM1.
PKM2 as a Transcriptional Co-Activator
PKM2 is also phosphorylated on serine residues at positions 37 (S37) and 202 (S202). Serine 37 phosphorylation of PKM2 stimulates its movement into the nucleus where it functions as a transcriptional co-activator of β-catenin in the activation of the MYC gene. MYC is a potent transcription factor and elevated expression of MYC contributes to the transformed state of cancer cells. In addition, several MYC target genes contribute to the increased metabolic profiles of cancer cells. These genes include the SLC2A1 gene that encodes glucose transporter 1 (GLUT1), the LDHA gene that encodes lactate dehydrogenase A, and also the PKM gene leading to increased levels of PKM2. This latter effect results in a positive feed-back loop between PKM2 and MYC.
Serine 202 phosphorylation also results in migration of PKM2 to the nucleus and this modification allows PKM2 to serve as a transcriptional co-activator of the transcription factor signal transducer and activator of transcription 5A (STAT5A) resulting enhanced transcription of a member of the cyclin family (cyclin D1) of cyclin-dependent kinase (CDK) regulators leading to enhanced cell cycle progression.
Phosphoglycerate Mutase in PKM2 Expressing Cells
In cells in culture, the replacement of PKM2 with PKM1 (the constitutively active isoform) results in reduced lactate production and enhanced oxygen consumption. An additional observation has been made that in cells expressing PKM2 there is increased phosphorylation of an active site histidine (His11) in the upstream glycolytic enzyme phosphoglycerate mutase (PGAM1). His11 phosphorylation of PGAM1 increases its mutase activity. The phosphorylation of PGAM1 is not seen in PKM1 expressing cells.
It turns out that 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 reaction occurs at physiological concentrations of PEP and produces pyruvate in the absence of the normal glycolytic pyruvate kinase activity. Thus, the PEP-dependent histidine phosphorylation of PGAM1 may provide an alternate glycolytic pathway that decouples ATP production from normal pyruvate kinase-mediated phosphotransfer from PEP. This alternate pathway allows for a high rate of glycolysis that is needed to support the anabolic metabolism observed in many proliferating cells. In addition to altered pyruvate kinase activity, cancer cells also have increased levels of expression of the kinases that phosphorylate the pyruvate dehydrogenase complex (PDHc), further limiting pyruvate oxidation, and increased expression of lactate dehydrogenase which drives pyruvate into lactate production (see the next section below).
PGAM1 is unique with respect to glycolytic enzymes because its rate of transcription is regulated by the tumor suppressor p53. In addition, increased expression of PGAM1 has been shown to immortalize primary cells, although the mechanism of this immortalization remains unknown. When PKM2 activity is down-regulated, as a consequence of growth factor-mediated tyrosine phosphorylations, PGAM1 mutase activity is enhanced due to the consequent increase in His11 phosphorylation from PEP. Thus, a positive feedback loop is activated, whereby the production of PEP increases the enzymatic activity of PGAM1. Activation of this feedback loop between PEP and His11 modified PGAM1 may be the mechanism that promotes the redistribution of glycolytic carbons, upstream of PGAM1, into biosynthetic pathways that branch from glycolysis. In order for this alternative pathway to continue, the phosphate on His11 of PGAM1 must be removed so that it can serve as a continual acceptor of PEP phosphate. When PGAM1 converts 3-phosphoglycerate (3-PG) to 2-phosphoglycerate (2-PG) there is spontaneous hydrolysis of the phosphohistidine. In addition, it has been observed that 2,3-bisphosphoglycerate (2,3-BPG) can be formed from either 3-PG or 2-PG via phosphate transfer from His11 of PGAM1.
PKM2 Benefit for Cancer Cell Proliferation
So, for proliferating cells such as cancers, even though it may seem counterproductive to prevent complete oxidation of glucose solely for ATP production, the demands for carbon incorporation into biomass clearly supersedes the needs for ATP production from glucose. Also, cancer cells bypass the hormonal signals required of normal cells for nutrient uptake so there is no limit to the sources of carbon atoms for ATP production (e.g. fatty acids and amino acids). Of metabolic significance to proliferating cells is that they must avoid ATP production in excess of demand to avoid allosteric inhibition of PFK-1 and other rate-limiting steps in glycolysis that are inhibited by a high ATP/ADP ratio. Therefore, the inhibition of PKM2 by binding to tyrosine phosphorylated proteins, following growth factor stimulation, may serve to uncouple the ability of cells to divert the carbons from nutrients (such as glucose) into biosynthetic pathways from the production of ATP. This may, in fact, be the underlying reason why PKM2 activity has evolved to be decreased in rapidly dividing cells.
PKM2 as Anti-Cancer Target
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.
Glucose Metabolism and Cancer: The Hypoxia Induced Pathway
As described in the previous section, altered metabolism of glucose is a hallmark of all types of cancer. The diversion of glucose carbons into biomass in cancer cells necessitates an increased delivery of glucose into these cells and an increase in the rate of anaerobic glycolysis to lactate. This is accomplished by an increase in the expression of genes encoding glucose transporters and glycolytic enzymes. These transcriptional changes can be observed in over 70% of human cancers and is driven in part through activation of the hypoxia induced factor 1 (HIF-1) pathway and by increased expression of various proto-oncogenes and decreased expression of various tumor suppressors.
The HIF-1 pathway, which is activated by conditions of hypoxia (low oxygen tension), is a major homeostatic mechanism for cellular responses to changes in the level of oxygen within cells. HIF-1 is a heterodimeric complex composed of an α-subunit and a β-subunit. The β-subunit is constitutively expressed while the α-subunit expression and activity are increased in response to changes in cellular oxygen content.
There are three related HIF complexes identified as HIF-1, HIF-2, and HIF-3 that are defined by the particular α-subunit of the complex. The activity of the HIF-1 and HIF-2 complexes are highly similar in their responses to hypoxia. Less detail is known regarding the HIF-3 complex.
Humans express three α-subunit genes, HIF1α (HIF1A gene), HIF2α (EPAS1 gene, for endothelial PAS domain protein 1), and HIF3α (HIF3A gene). The PAS domain is so-called because of the three proteins in which the domain was originally identified: Per (period circadian protein), ARNT (aryl hydrocarbon receptor nuclear translocator), and Sim (simple-minded protein).
The original β-subunit (HIF1β) was initially identified and characterized as ARNT. Humans express two ARNT-related genes (ARNT2 and ARNTL), however, the encoded proteins are not components of the HIF complexes.
Normally the HIF1α subunits are degraded in the presence of oxygen due to polyubiquitylation. Polyubiquitylation is a key modification directing proteins for rapid degradation by the proteasome machinery.
Expression of the HIF1A gene is ubiquitous, whereas expression of the EPAS1 (HIF2A) gene is more restricted being primarily found active in interstitial cells, endothelial cells, and parenchymal cells. Expression of the HIF3A gene is found in numerous tissues with highest levels in adipose tissue, heart, spleen, and placenta.
The HIF α-subunits possess an oxygen-dependent degradation (ODD) domain. The ODD domain is hydroxylated by a member of the prolyl hydroxylase domain (PHD) family of proline hydroxylating enzymes. The prolyl hydroxylase family includes the enzymes that incorporate hydroxyl groups into proline residues in collagens of the extracellular matrix. The prolyl hydroxylases that hydroxylate HIF α-subunits are all ferrous (Fe2+) iron and 2-oxoglutarate (α-ketoglutarate)-dependent enzymes. The requirement of these enzymes for 2-oxoglutarate results in direct coupling of the activity this class of prolyl hydroxylases to metabolic processes that generate and utilize 2-oxoglutarate such as the TCA cycle.
The 2-oxoglutarate-dependent prolyl hydroxylase enzymes are identified as PHD1 (encoded by the EGLN2 gene), PHD2 (encoded by the EGLN1 gene) and PHD3 (encoded by the EGLN3 gene). The designation of EGLN refers to the fact that these three genes are homologs of the Caenorhabditis elegans egg laying-9 (Egl-9) gene. Hydroxylation of HIF α-subunits renders the proteins susceptible to proteasomal degradation under normoxic cellular conditions. In response to proline hydroxylation the ubiquitin ligase encoded by the von Hippel-Lindau (VHL) gene binds to the HIF α-subunit proteins and catalyzes their polyubiquitylation.
Expression of PHD1 is highest in the testes with lower level expression seen in brain, liver, kidney, and heart. Expression of PHD2 is observed in most tissues. Expression of PHD3 is highest within cardiac myocytes. The activities of PHD2 and PHD3 are strongly induced by changes in oxygen concentrations.
The hydroxylation reactions catalyzed by the PHD enzymes require molecular oxygen (O2) in addition to the Fe2+ and 2-oxoglutarate, therefore, reductions in oxygen content will result in loss of their activity. The products of the PHD enzymes are a trans-4-hydroxyproline residues, CO2, and succinate. As indicated, expression of the HIF1A gene is ubiquitous and this pattern is maintained under normal oxygen availability (normoxic conditions).
The activity of the PHD enzymes is not only regulated by oxygen concentration but by numerous metabolites as well as by reactive oxygen species, ROS. ROS inhibit PDH activity allowing for stabilization of HIF1a and its migration to the nucleus. Similarly, metabolites that include pyruvate, lactate, succinate, and fumarate inhibit the activity of PDH enzymes. The significance of the metabolic inhibition of PDH activity relates to the altered metabolic profile of cancer cells and how this altered metabolism aids in the propagation of cancer cells, in part, through inhibition of HIF1a degradation. In addition to the normal metabolites that inhibit PDH enzymes, 2-hydroxyglutarate, that is formed as a result of the altered activity of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) resulting from mutations in these genes, also inhibits the PDH enzymes. This latter process contributes to the cancers that develop in individuals harboring mutant IDH1 or IDH2 genes.
The activity of the HIF1α protein is regulated by being hydroxylated on two prolines (P405 and P531) in the ODD. The presence of the trans-4-hydroxyproline residues increases the binding of the VHL encoded protein by over 1000 fold. Given that the PHD enzymes require O2 as a substrate for the hydroxylation reaction, when conditions of hypoxia exist the HIFα subunits escape hydroxylation and are, therefore, not ubiquitylated. Under hypoxic conditions the stabilized HIFα subunits migrate to the nucleus and dimerize with the HIFβ subunit and activate the expression of target genes.
The activity of HIF1α is also regulated via the hydroxylation of a specific asparagine residue (N803) found in the C-terminal transactivation domain. The N803 hydroxylation is catalyzed by another 2-oxoglutarate-dependent dioxygenase originally identified as factor-inhibiting HIF-1 (FIH1; also identified as FIH). FIH1 is encoded by the HIF1AN (hypoxia inducible factor 1 alpha subunit inhibitor) gene. The consequences of the β-hydroxylation of N803 are that HIF1α can no longer interact with the transcriptional co-activators CBP [cAMP-response element-binding protein (CREB)- binding protein] and p300 (CBP/p300) resulting in inhibition of HIF-1 activity.
Both HIF1α and HIF1β proteins are transcription factors that contain basic helix-loop-helix domains that allow them to dimerize and to bind to DNA sequences in target genes. Both HIF1α and HIF1β also have a PAS domain. The HIF1α subunits also contain two transactivation domains (TAD), which regulate the expression of HIF-1 target genes. As indicated, the transcriptional coactivator proteins CBP and p300 (CBP/p300) interact with HIF1α and this interaction occurs through the C-terminal TAD. CBP and p300 modify chromatin structure, and thereby transcriptional activity, via their lysine acetyltransferase (KAT) activity which acetylates the nucleosomal histones.
The normal role of the HIF-1 pathway is to promote the delivery of oxygen and nutrients to the oxygen-deprived tissue via the stimulation of neovascularization (angiogenesis). The microenvironment that surrounds most solid tumors is highly hypoxic and, therefore, the ability of the tumor cells to proliferate requires the ability to acquire oxygen and nutrients. This is accomplished, in large part, through the activation of the HIF-1 pathway which is considered to be a modulator in the transactivation of genes implicated in the altered metabolism observed in cancer cells.
Several of the metabolic regulatory genes that are activated by HIF-1 include the pyruvate kinase M (PKM) gene, described in detail in the previous section, the fructose-1,6-bisphosphate aldolase (ALDOA) gene, the pyruvate dehydrogenase kinase 1 (PDK1) gene, the GLUT1 gene, and the lactate dehydrogenase A (LDHA) gene. The altered patterns of metabolism lead to the accumulation of diverse metabolites in the microenvironment that promote tumor growth and contribute to the ability of the tumor cells to metastasize. Activation of HIF-1 in cancer cells results in restriction of glucose entry into the mitochondrial oxidative phosphorylation pathway via inhibition of the pyruvate dehydrogenase complex, PDHc. The inhibition of the PDHc is effected via HIF-1 stimulated expression of the PDK1 gene. The regulation of the PDHc by phosphorylation is discussed in detail in the TCA Cycle page.
The altered pyruvate kinase isoforms, as occurs in cancer (see previous section), coupled with the activation of the HIF-1 pathway results in diversion of glucose metabolism into the pentose phosphate pathway as well as into increased lactate production. The conversion of pyruvate into lactate is enhanced in the context of activated HIF-1 since this transcription factor activates the expression of the LDHA gene. The increased production of lactate, by cancer cells, contributes to the acidification of the tumor microenvironment which, in turn, promote further activation of the HIF-1 pathway. Lactate accumulation also results in pyruvate accumulation in cancer cells. Pyruvate is a known inhibitor of the prolyl hydroxylases that hydroxylate the HIF1α subunit proteins. Loss of HIF1α proline hydroxylation results in increased HIF1α stability and, therefore, increased HIF-1 transcriptional activity. Thus, accumulation of lactate and pyruvate, which occurs as a result of both altered pyruvate kinase gene expression and activation of the HIF-1 pathway, further promotes activation of the HIF-1 pathway leading to a controlled and enhanced metabolic profile within cancer cells.
The Glucose-Fatty Acid Cycle
The glucose-fatty acid cycle describes interrelationships of glucose and fatty acid oxidation as defined by fuel flux and fuel selection by various organs. This cycle is not a metabolic cycle such as can be defined by the TCA cycle as an example, but defines the dynamic interactions between these two major energy substrate pools. The glucose-fatty acid cycle was first proposed by Philip Randle and co-workers in 1963 and is, therefore, sometimes referred to as the Randle cycle or Randle hypothesis. The cycle describes how nutrients in the diet can fine-tune metabolic processes on top of the more coarse control exerted by various peptide and steroid hormones. The underlying theme of the glucose-fatty acid cycle is that the utilization of one nutrient (e.g. glucose) directly inhibits the use of the other (in this case fatty acids) without hormonal mediation. The general interrelationships between glucose and fatty acid utilization in skeletal muscle and adipose tissue that constitutes the glucose-fatty acid cycle are diagrammed in the Figure below.
How do the dynamics of the glucose-fatty acid cycle play out under various physiological conditions and changing fuel substrate pools? In the fasted state it is imperative that glucose be spared so that the brain can have adequate access to this vital fuel. Under these conditions, hormonal signals from the pancreas, in the form of glucagon, stimulate adipose tissue lipolysis releasing free fatty acids (FFAs) to the blood for use as a fuel by other peripheral tissues. When the released FFAs enter the liver they oxidized and also serve as substrates for ketogenesis. The oxidation of fatty acids inhibits glucose oxidation as outlined in the above figure. In addition to sparing glucose for the brain, fatty acid oxidation also preserves pyruvate and lactate which are important gluconeogenesis substrates. The effects of fatty acids on glucose utilization can also be observed in the well fed state after a high fat meal and during periods of exercise.
As outlined in the above Figure, the inhibition of glucose utilization by fatty acid oxidation is mediated by short-term effects on several steps of overall glycolysis that include glucose uptake, glucose phosphorylation and pyruvate oxidation. During fatty acid oxidation the resultant acetyl-CoA allosterically activates PDKs that phosphorylate and inhibit the PDHc. PDKs are also activated by increasing levels of NADH that will be the result of increased fatty acid oxidation. Thus, two products of fat oxidation result in inhibition of the PDHc. In addition, excess acetyl-CoA is transported to the cytosol either as citrate (as diagrammed) or as acetylcarnitine. Mitochondrial acetylcarnitine is formed through the action of carnitine acetyltransferase (CAT). Acetylcarnitine is transported out of the the mitochondria via the action of carnitine-acylcarnitine translocase (CACT: SLC25A20). Once in the cytosol acetylcarnitine is converted to acetyl-CoA via the action of cytosolic CAT. In the cytosol, citrate serves as an allosteric inhibitor of PFK1 thus limiting entry of glucose into glycolysis. The increase in glucose-6-phosphate that results from inhibition of PFK1 leads to feed-back inhibition of hexokinase which in turn limits glucose uptake via GLUT4. Additional mechanisms of fatty acid metabolism that lead to interference in glucose uptake and utilization are the result of impaired insulin receptor signaling. These latter processes are discussed in detail in the Insulin Function page.
Mechanisms by which glucose utilization inhibits fatty acid oxidation are tissue specific due primarily to the differences in Km of hepatic glucokinase and skeletal muscle and adipose tissue hexokinase. In addition, hepatic CPT-1 is approximately 100-fold less sensitive to inhibition by malonyl-CoA than are the skeletal muscle and cardiac isoforms. When glucose is oxidized in glycolysis the resultant pyruvate enters the mitochondria via the pyruvate symporter. Increasing mitochondrial pyruvate inhibits the PDKs allowing for rapid decarboxylation of pyruvate by the PDHc ensuring continued entry of glucose into the glycolytic stream. Some of the acetyl-CoA derived from pyruvate oxidation will be diverted from the TCA cycle as citrate and transported to the cytosol by the tricarboxylic acid transporter (TCAT). The citrate is converted to acetyl-CoA and oxaloacetate by ATP-citrate lyase (ACLY) and can now serve as a substrate for ACC. The resultant malonyl-CoA will inhibit CPT-1 thus, restricting mitochondrial uptake and oxidation of fatty acyl-CoAs. The inhibition of fatty acid oxidation in the liver re-routes LCFAs into triglycerides (TAGs). Long term effects of excess glucose are reflected in hepatic steatosis resulting from the diversion of fats into TAGs instead of being oxidized.
In addition to being regulated by intermediates of glucose and fat oxidation, several enzymes in these two pathways are regulated at the level of post-translational modification and/or gene expression. Most of these regulatory schemes have been covered in the above sections or in the Fatty Acid Oxidation page.
Metabolic Fates of Pyruvate
Pyruvate is the branch point molecule of glycolysis. The ultimate fate of pyruvate depends on the oxidation state of the cell. In the reaction catalyzed by GAPDH a molecule of NAD+ is reduced to NADH. In order to maintain the redox state of the cell, this NADH must be re-oxidized to NAD+. During aerobic glycolysis this occurs in the mitochondrial electron transport chain generating ATP. Thus, during aerobic glycolysis ATP is generated from oxidation of glucose directly at the PGK and PK reactions as well as indirectly by re-oxidation of NADH in the oxidative phosphorylation pathway. Additional NADH molecules are generated during the complete aerobic oxidation of pyruvate in the TCA cycle. Pyruvate enters the TCA cycle in the form of acetyl-CoA which is the product of the pyruvate dehydrogenase reaction. The fate of pyruvate during anaerobic glycolysis is reduction to lactate.
During anaerobic glycolysis, that period of time when glycolysis is proceeding at a high rate (or in anaerobic organisms), 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. If the energy level in the liver is high, the carbons of pyruvate will be diverted back to glucose via the gluconeogenesis pathway.
There are two distinct forms of LDH determined by their specificity toward L-lactate and/or D-lactate. These enzymes are encoded for by four different genes in humans identified as LDHA, LDHB, LDHC, and LDHD. Only the LDHD encoded enzyme shows specificity for D-lactate. The LDHA gene encodes the muscle-specific (M) subunit of LDH. The LDHB gene encodes the heart-specific (H) subunit of LDH. As indicated below, different combinations of the M and H subunits generates LDH isoforms in different tissues. The protein encoded by the LDHC gene is found only in the testis. The enzyme encoded by the LDHD gene is a mitochondria-specific enzyme whose expression appears to rise in certain types of cancer (e.g. prostate cancers).
The LDHA gene is located on chromosome 11p15.1 and is composed of 9 exons that generate multiple alternatively spliced mRNAs.
The LDHB gene is located on chromosome 12p12.1 and is composed of 8 exons that generate two alternatively spliced mRNAs that both encode the same 334 amino acid protein. In addition to the 334 amino acid protein, one of the alternatively spliced LDHB mRNAs encodes a distinct 341 amino acid protein identified as LDHBx. The LDHBx protein results from translational read-through of the normal UGA stop codon with termination occurring at a downstream UAG codon. Analysis of the LDHBx protein demonstrates that several different amino acids (Arg, Cys, Ser, or Trp) can be found at the position of the UGA codon. The LDHBx protein localizes to the peroxisomes.
The LDHC gene is located on chromosome 11p15.1 and is composed of 8 exons that generate two alternatively spliced mRNAs that both encode the same 332 amino acid protein.
The LDHD gene is located on chromosome 16q23.1 and is composed of 11 exons that generate two alternatively spliced mRNAs encoding two isoforms of this enzyme. Mutations in the LDHA gene are associated with the glycogen storage disease type 11, GSD11.
The majority of the functional LDH in mammalian cells contain various combinations two distinct types of LDH subunits, termed M ( encoded by the LDHA gene) and H (encoded by the LDHB gene). 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 also is 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 is utilized to reduce pyruvate to lactate. The various other isoforms of LDH are described in the Enzyme Kinetics page.
Glucuronate is a highly polar molecule which is incorporated into proteoglycans as well as combining with bilirubin and steroid hormones; it can also be combined with certain drugs to increase their solubility. Glucuronate is derived from glucose in the uronic acid pathway.
The uronic acid pathway is an alternative pathway for the oxidation of glucose that does not provide a means of producing ATP, but is utilized for the generation of the activated form of glucuronate, UDP-glucuronate. The uronic acid pathway of glucose conversion to glucuronate begins by conversion of glucose-6-phosphate to glucose-1-phosphate by phosphoglucomutase, and then activated to UDP-glucose by UDP-glucose pyrophosphorylase. UDP-glucose is oxidized to UDP-glucuronate by the NAD+-requiring enzyme, UDP-glucose dehydrogenase (encoded by the UGDH gene). UDP-glucuronate then serves as a precursor for the synthesis of iduronic acid and UDP-xylose and is incorporated into proteoglycans and glycoproteins or forms conjugates with bilirubin, steroids, xenobiotics, drugs and many compounds containing hydroxyl (–OH) groups.
Clinical Significance of Glucuronate
In the adult human, a significant number of erythrocytes die each day. This turnover releases significant amounts of the iron-free portion of heme, porphyrin, which is subsequently degraded. The primary sites of porphyrin degradation are found in the reticuloendothelial cells of the liver, spleen and bone marrow. The breakdown of porphyrin yields bilirubin, a product that is non-polar and therefore, insoluble. In the liver, to which is transported in the plasma bound to albumin, bilirubin is solubilized by conjugation to glucuronate. The soluble conjugated bilirubin diglucuronide is then secreted into the bile. An inability to conjugate bilirubin, for instance in hepatic disease or when the level of bilirubin production exceeds the capacity of the liver, is a contributory cause of jaundice.
The conjugation of glucuronate to certain non-polar drugs is important for their solubilization in the liver. There are numerous genes in the human genome that encode UDP-glucuronyltransferases which themselves belong to the large superfamily of glycosyltransferases. There are four UDP-glucuronyltransferase families in humans identified as the UGT1, UGT2, UGT3, and UGT8 families. There are nine functional enzymes of the UGT1 family that are derived through the splicing of nine alternative first exons to the four common exons (2, 3, 4, and 5). These enzymes are designated UGT1A1–UGT1A9 (described in more detail in the Heme Metabolism page). The UGT2 gene family is composed of three UGT2A members (UGT2A1, UGT2A2, and UGT2A3) and seven UGT2B members (UGT2B,4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28). The UGT3 family is composed of two members (UGT3A1 and UGT3A2) and the UGT8 family is composed of a single member (UGT8A1). Although the preferred substrate for these enzymes is UDP-glucuronate as the glycosyl donor, other UDP sugars (e.g. UDP-glucose and UDP-xylulose) may be used. Whereas most all of these enzymes catalyze reactions designed to detoxify both endogenous and exogenous compounds, the UGT8A1 enzyme (identified as UDP-galactose ceramide galactosyltransferase and abbreviated CGT) carries out a biosynthetic reaction in galactocerebroside synthesis.
Glucuronate-conjugated drugs are more easily cleared from the blood by the kidneys for excretion in the urine. The glucuronate-drug conjugation system can, however, lead to drug resistance; chronic exposure to certain drugs, such as barbiturates and AZT, leads to an increase in the synthesis of the UDP-glucuronyltransferases in the liver that are involved in glucuronate-drug conjugation. The increased levels of these hepatic enzymes result in a higher rate of drug clearance leading to a reduction in the effective dose of glucuronate-cleared drugs.
Regulation of Blood Glucose Levels
If for no other reason, it is because of the demands of the brain for oxidizable glucose that the human body exquisitely regulates the level of glucose circulating in the blood. This level is maintained in the range of 5mM (90mg/dL) during normal between meal fasting.
Nearly all carbohydrates ingested in the diet are converted to glucose following transport to the liver. Catabolism of dietary or cellular proteins generates carbon atoms that can be utilized for glucose synthesis via gluconeogenesis. Additionally, other tissues besides the liver that incompletely oxidize glucose (predominantly skeletal muscle and erythrocytes) provide lactate that can be converted to glucose via gluconeogenesis.
Maintenance of blood glucose homeostasis is of paramount importance to the survival of the human organism. The predominant tissue responding to signals that indicate reduced or elevated blood glucose levels is the liver. Indeed, one of the most important functions of the liver is to produce glucose for the circulation. Both elevated and reduced levels of blood glucose trigger hormonal responses to initiate pathways designed to restore glucose homeostasis. Low blood glucose triggers release of glucagon from pancreatic α-cells. High blood glucose triggers release of insulin from pancreatic β-cells.
Additional hormonal signals, such as via ACTH and growth hormone, released from the pituitary, act to increase blood glucose by inhibiting its uptake by extrahepatic tissues such as adipose tissue and skeletal muscle. Glucocorticoids also act to increase blood glucose levels by inhibiting glucose uptake (also primarily at the level of adipose tissue and skeletal muscle) and by stimulation of gluconeogenesis. Cortisol, the major glucocorticoid released from the adrenal cortex, is secreted in response to the increase in circulating ACTH. Within the liver, cortisol binding to the glucocorticoid receptor (GR), results in transcriptional activation of the PEPCK gene, thereby, resulting in increased rates of gluconeogenesis and glucose output to the blood.
The adrenal medullary hormone, epinephrine, stimulates production of glucose by activating hepatic glycogenolysis and gluconeogenesis. These effects are exerted via the presence of α1 and β2 adrenergic receptor subtypes on hepatocytes. Epinephrine also exerts an effect on skeletal muscle glycogenolysis in response to stressful stimuli. Within skeletal muscle, epinephrine exerts its effects primarily through activation of the β2 adrenergic receptor but a small percentage of the total adrenergic receptor subtypes in skeletal muscle includes the β1 subtype (7–10% of the total).
The significance of adrenergic (epinephrine primarily) receptor function to the control of blood glucose can be seen by the consequences of several identified receptor mutations. For example, mutations in either the β1 or the β3 adrenergic receptors are highly correlated to insulin resistance associated with type 2 diabetes. In addition, mutations in all three β-adrenergic receptors are associated with hyperlipidemia (which exacerbates the hyperglycemia of diabetes) as well as the associated pathophysiology of the metabolic syndrome. Mutations in all three β-receptors are also associated with increased risk for obesity.
Glucagon binding to its receptors on the surface of liver cells triggers an increase in cAMP production leading to an increased rate of glycogenolysis by activating glycogen phosphorylase via the PKA-mediated cascade. This is the same response hepatocytes have to epinephrine binding to the β2 adrenergic receptors on hepatocytes. The resultant increased levels of G6P in hepatocytes is hydrolyzed to free glucose, by glucose-6-phosphatase, which then diffuses to the blood. The glucose enters extrahepatic cells where it is re-phosphorylated by hexokinase. Since all tissues, excluding liver, kidney, and small intestine, lack glucose-6-phosphatase, the glucose-6-phosphate product of hexokinase is retained and oxidized by these tissues.
In opposition to the cellular responses to glucagon, cortisol, and epinephrine, insulin stimulates extrahepatic uptake of glucose from the blood and inhibits glycogenolysis in extrahepatic cells and conversely stimulates glycogen synthesis. As the glucose enters hepatocytes it binds to and inhibits glycogen phosphorylase activity. The binding of free glucose stimulates the dephosphorylation of phosphorylase thereby, inactivating it. Why is it that the glucose that enters hepatocytes is not immediately phosphorylated and oxidized? Hepatocytes express the isoform of hexokinase called glucokinase. Glucokinase has a much lower affinity for glucose than does hexokinase. Therefore, it is not fully active at the physiological ranges of blood glucose. Additionally, glucokinase is not inhibited by its product G6P, whereas, hexokinase is inhibited by G6P.
Hepatocytes, unlike most other cells, are essentially freely permeable to glucose and are, therefore, not directly affected by the action of insulin at the level of increased glucose uptake. When blood glucose levels are low, the liver does not compete with other tissues for glucose since the extrahepatic uptake of glucose is stimulated in response to insulin. Conversely, when blood glucose levels are high extrahepatic needs are satisfied and the liver takes up glucose for conversion into glycogen for future needs. Under conditions of high blood glucose, liver glucose levels will be high and the activity of glucokinase will be elevated. The G6P produced by glucokinase is rapidly converted to G1P by phosphoglucomutase, where it can then be incorporated into glycogen.
Role of the Kidney in Blood Glucose Control
Although the liver is the major site of glucose homeostasis, the kidney plays a vital role in the overall process of regulating the level of blood glucose. The kidney carries out gluconeogenesis primarily using the carbon skeleton of glutamine and while so doing allows for the elimination of waste nitrogen and maintaining plasma pH balance. For the role of the kidneys in gluconeogenesis please visit that section of the Gluconeogenesis page. In addition to carrying out gluconeogenesis, the kidney regulates blood glucose levels via its ability to excrete glucose via glomerular filtration as well as to reabsorb the filtered glucose in the proximal convoluted tubules. In the average adult the kidneys will filter around 180gm of glucose per day. Of this amount less than 1% is excreted in the urine due to efficient reabsorption. This reabsorption process is critical for maintaining blood glucose homeostasis and for retaining important calories for energy production.
Transport of glucose from the tubule into the tubular glomerular epithelial cells is carried out by specialized transport proteins termed sodium-glucose co-transporters (SGLTs). The SGLTs represent a family of transporters that are involved in the transport of glucose, amino acids, vitamins, and ions and other osmolytes across the brush-border (apical) membranes of kidney tubule cells and intestinal epithelial cells. There are two SGLT transporters in the kidney involved in glucose reabsorption. SGLT1 is found primarily in the distal S3 segment of the proximal tubule and SGLT2 is expressed in the S1 and S2 segments (see the Figure below). The location of SGLT2 in the proximal tubule means that it is primarily responsible for glucose reabsorption. SGLT2 is a high-capacity low-affinity transporter that, due to its expression location, is responsible for approximately 90% of the glucose reabsorption activity of the kidney.
As would be expected from the name of the renal glucose transporters, SGLT1 and SGLT2 catalyze the active transport of glucose against a concentration gradient across the lumenal (apical) membrane of the tubule cell and couple this transport to sodium uptake. The inward sodium uptake is maintained by ATP-driven active transport of the sodium across the basolateral membrane into the blood (coupled to inward uptake of potassium) via the activity of the renal Na+,K+-ATPase. The reabsorbed glucose passively diffuses out of the tubule cell into the blood via the basolateral membrane associated GLUT2. Under normal conditions saturation of the ability of SGLT2 to reabsorb glucose is never reached. The kidney can filter and reabsorb approximately 375mg of glucose per minute. The plasma concentration of glucose required to exceed this capacity is well above that considered normal and is only observed in situations of renal dysfunction/disease or most importantly in type 2 diabetes. Because of the importance of SGLT2 in renal reabsorption of glucose this transporter has become the target for therapeutic intervention of the hyperglycemia associated with type 2 diabetes. By specifically inhibiting SGLT2 there will be increased glucose excretion in the urine and thus, a lowering of plasma glucose levels. Several SGLT2-specific inhibitors have recently been approved for use in the treatment of the hyperglycemia of type 2 diabetes. All of the names of the drugs in the SGLT2 inhibitor class have the suffix, -gliflozin. For information on the SGLT2 inhibitors visit the Diabetes page.
One major response of non-hepatic tissues to insulin is the recruitment, to the cell surface, of glucose transporter complexes. 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.
There are two additional glucose transporters which are the Na+-dependent glucose transporters, SGLT1 and SGLT2 (see section above). The SGLT acronym refers to sodium-glucose linked transporter. Both SGLT1 and SGLT2 are members of the solute carrier 5 family, thus the gene encoding SGLT1 is SLC5A1 and that encoding SGLT2 is SLC5A2. These transporters are expressed in the enterocytes of the small intestine and within epithelial cells of the proximal tubules of the kidney. Within the small intestine SGLT1 contributes to dietary glucose and galactose absorption as described earlier. Within the kidney, SGLT1 is expressed in the S3 segment of the tubule and SGLT2 is expressed in the S1 and S2 segments. The activity of renal SGLT2 accounts for over 90% of the glucose reabsorption by the kidney. Due to this activity, this transporter has become a pharmacologic target for the treatment of the hyperglycemia associated with type 2 diabetes.
The GLUT family of glucose transporters can be divided into three classes based upon primary amino acid sequence comparisons. Class I transporters include GLUT1, GLUT2, GLUT3 (and the gene duplication of GLUT3 identified as GLUT14), and GLUT4. Class II transporters include GLUT5, GLUT7, GLUT9 and GLUT11. Class III transporters include 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 brain, placenta, and erythrocytes. In fact in erythrocytes GLUT1 accounts for almost 5% of total protein. Although widely expressed, GLUT1 is not expressed in hepatocytes. GLUT1 is the primary transporter responsible for glucose transport across the blood-brain-barrier. Deficiencies in GLUT1 results in GLUT1 deficiency syndrome.
GLUT2 is found primarily in intestine, pancreatic β-cells, kidney and liver. The Km of GLUT2 for glucose (17mM) 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 but also found 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.
Insulin-sensitive tissues, such as skeletal muscle and adipose tissue, contain GLUT4 whose mobilization to the cell-surface is stimulated by insulin action.
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.
GLUT9 (SLC2A9) does not transport sugar but is a uric acid transporter abundant in the kidney and liver.
Recent evidence has shown that one of the cell surface binding sites for the human T cell leukemia virus (HTLV) is the ubiquitous GLUT1.