Glycolysis: Process of Glucose Utilization and Homeostasis


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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 simpler, soluble 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 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 maltases that hydrolyze di- and trisaccharides, and the more specific disaccharidases, sucrase-isomaltase, lactase, and trehalase. The net result is the almost complete conversion of digestible carbohydrate to its constituent monosaccharides. The resultant glucose and other simple carbohydrates are transported across the intestinal wall to the hepatic portal vein and then to liver parenchymal cells and other tissues. There they are converted to fatty acids, amino acids, and glycogen, or else oxidized by the various catabolic pathways of cells.

Oxidation of glucose is known as glycolysis.Glucose is oxidized to either lactate or pyruvate. 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.

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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+

Reactions of glycolysis

Pathway of glycolysis from glucose to pyruvate: A larger vertical Figure is included in the Individual Reactions of Glycolysis section below. In this larger image one can mouse over structure names to see the chemical structures of the intermediates. The enzyme abbreviations are also identified in the Figure below.


The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative phosphorylation, producing either two or three equivalents 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.

Malate-aspartate shuttle

The malate/aspartate shuttle is the principal mechanism for the movement of reducing equivalents (in the form of NADH; highlighted in the red boxes) from the cytoplasm to the mitochondria. The glycolytic pathway is a primary source of NADH. Within the mitochodria the electrons of NADH can be coupled to ATP production during the process of oxidative phosphorylation. The electrons are "carried" into the mitochondria in the form of malate. Cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD+. Malate then enters the mitochondria where the reverse reaction is carried out by mitochondrial MDH. Movement of mitochondrial OAA to the cytoplasm to maintain this cycle requires it be transaminated to aspartate (Asp, D) with the amino group being donated by glutamate (Glu, E). The Asp then leaves the mitochondria and enters the cytoplasm. The deamination of glutamate generates α-ketoglutarate (α-KG) which leaves the mitochondria for the cytoplasm. All the participants in the cycle are present in the proper cellular compartment for the shuttle to function due to concentration dependent movement. When the energy level of the cell rises the rate of mitochondrial oxidation of NADH to NAD+ declines and therefore, the shuttle slows. GAPDH is glyceraldehyde-3-phosphate dehydrogenase. AST is aspartate transaminase. SLC25A11 is the malate transporter and SLC25A13 is the aspartate/glutamate transporter.


Glycerol phopshate shuttle

The glycerol phosphate shuttle is a secondary mechanism for the transport of electrons from cytosolic NADH to mitochondrial carriers of the oxidative phosphorylation pathway. The primary cytoplasmic NADH electron shuttle is the malate-aspartate shuttle (see below). Two enzymes are involved in this shuttle. One is the cytosolic version of the enzyme glycerol-3-phosphate dehydrogenase (GPD1) which has as one substrate, NADH. The second is is the mitochondrial form (GPD2) of the enzyme which has as one of its' substrates, FAD+. The net result is that there is a continual conversion of the glycolytic intermediate, DHAP and glycerol-3-phosphate with the concomitant transfer of the electrons from reduced cytosolic NADH to mitochondrial oxidized FAD+. Since the electrons from mitochondrial FADH2 feed into the oxidative phosphorylation pathway at coenzyme Q (as opposed to NADH-ubiquinone oxidoreductase [complex I]) only 2 moles of ATP will be generated from glycolysis. GAPDH is glyceraldehyde-3-phoshate dehydrogenase.


The net yield from the oxidation of 1 mole of glucose to 2 moles of pyruvate is, therefore, either 6 or 8 moles of ATP. Complete oxidation of the 2 moles of pyruvate, through the TCA cycle, yields an additional 30 moles of ATP; the total yield, therefore being either 36 or 38 moles of ATP from the complete oxidation of 1 mole of glucose to CO2 and H2O.

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The Individual Reactions of Glycolysis

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

Reactions of glycolysis

Pathway of glycolysis from glucose to pyruvate: The two high energy intermediates whose oxidations are coupled to ATP synthesis are shown in red (1,3-bisphosphoglycerate and phosphoenolpyruvate). PGI: glucose-6-phosphate isomerase. TMI: triose isomerase. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. PGK1: phosphoglycerate kinase 1. PGAM1: phosphoglycerate mutase 1. Place mouse over intermediate names to see chemical structures.


The Hexokinase Reaction:

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

Four mammalian isozymes of hexokinase are known (Types I–IV), with the Type IV isozyme often referred to as glucokinase. Glucokinase is the form of the enzyme found in hepatocytes and pancreatic β-cells. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate.

Saturation curves comparing hexokinase and glucokinase

Comparison of the activities of hexokinase and glucokinase. The Km for hexokinase is significantly lower (0.1mM) than that of glucokinase (10mM). This difference ensures that non-hepatic tissues (which contain hexokinase) rapidly and efficiently trap blood glucose within their cells by converting it to glucose-6-phosphate. One major function of the liver is to deliver glucose to the blood and this in ensured by having a glucose phosphorylating enzyme (glucokinase) whose Km for glucose is sufficiently higher that the normal circulating concentration of glucose (5mM).

This feature 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 I, II, and III are allosterically inhibited by product (G6P) accumulation, whereas glucokinase is not. The 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. Although not product inhibited, hepatic glucokinase is allosterically inhibited by long-chain fatty acids (LCFA). In contrast, LCFAs do not inhibit the other forms of hexokinase. The ability of LCFAs to inhibit hepatic glucokinase is one of the mechanisms by which fatty acids inhibit glucose uptake into the liver. The inhibition of hepatic glucose uptake by LCFA is responsible, in part, for the hyperglycemia observed in obesity.


Glucose-6-phosphate isomerase:

The second reaction of glycolysis is an isomerization, in which G6P is converted to fructose 6-phosphate (F6P). The enzyme catalyzing this reaction is 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.


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, 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). Fibroblasts also express the PFKP gene. 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.


Aldolase:

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


Triose Phosphate Isomerase:

The two products of the aldolase reaction equilibrate readily in a reaction catalyzed by triose phosphate isomerase. Succeeding reactions of glycolysis utilize G3P as a substrate; thus, the aldolase reaction is pulled in the glycolytic direction by mass action principals.


Glyceraldehyde-3-Phosphate Dehydrogenase:

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


Phosphoglycerate Kinase:

The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase. Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal cell conditions. Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3-bisphosphoglycerate, 2,3BPG (see Figure below) by the enzyme bisphosphoglycerate mutase. 2,3BPG is an important regulator of hemoglobins' affinity for oxygen. Note that 2,3-bisphosphoglycerate phosphatase degrades 2,3BPG to 3-phosphoglycerate, a normal intermediate of glycolysis. The 2,3BPG shunt thus operates with the expenditure of 1 equivalent of ATP per triose passed through the shunt. The process is not reversible under physiological conditions.

Pathway for 2,3-bisphosphoglycerate synthesis in erythrocytes

The pathway for 2,3-bisphosphoglycerate (2,3-BPG) synthesis within erythrocytes. Synthesis of 2,3-BPG represents a major reaction pathway for the consumption of glucose in erythrocytes. The synthesis of 2,3-BPG in erythrocytes is critical for controlling hemoglobin affinity for oxygen. Note that when glucose is oxidized by this pathway the erythrocyte loses the ability to gain 2 moles of ATP from glycolytic oxidation of 1,3-BPG to 3-phosphoglycerate via the phosphoglycerate kinase reaction.


Phosphoglycerate Mutase and Enolase:

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 2PG by phosphoglycerate mutase and the 2PG conversion to phosphoenoylpyruvate (PEP) is catalyzed by enolase.


Pyruvate Kinase:

The final reaction of aerobic glycolysis is catalyzed by the highly regulated enzyme pyruvate kinase (PK). In this strongly exergonic reaction, the high-energy phosphate of PEP is 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 PK activity. One is located on chromosome 1, identified as the PKLR gene, and it encodes the liver (PKL or L-PK) and erythrocyte (PLR 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. Deficiencies in the PKLR gene are the cause of the most common form of inherited non-spherocytic anemia.

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Anaerobic Glycolysis

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.

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Regulation of Glycolysis

The reactions catalyzed by hexokinase, PFK-1 and PK all proceed with a relatively large free energy decrease. These non-equilibrium 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.

Regulation of hexokinase, however, is not the major control point in glycolysis. This is due to the fact that large amounts of G6P are derived from the breakdown of glycogen (the predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and, therefore, the hexokinase reaction is not necessary. Regulation of PK is important for reversing glycolysis when ATP is high in order to activate gluconeogenesis. As such this enzyme catalyzed reaction is not a major control point in glycolysis. The rate limiting step in glycolysis is the reaction catalyzed 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 PFK-1's ability 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.

Regulation of glycolysis and gluconeogenesis by fructose-2,6-bisphosphate

Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP). The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 is the kinase activity and F-2,6-BPase is the phosphatase activity of the bi-functional regulatory enzyme, phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2). PKA is cAMP-dependent protein kinase which phosphorylates PFK-2/F-2,6-BPase turning on the phosphatase activity. Green arrows represent positive actions. Red T-lines represent inhibitory actions.


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 inhibited. 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 if that targeting the enzyme for inhibition of the glycosylation 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 by a different gene that expresses several isoforms of each isozyme. The four different isozymes 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), encodes the liver isozyme. The PFKFB2 gene is found on chromosome 1q31 and encodes the heart isozyme. The PFKFB3 gene is located on chromosome 10p15–p14 and encodes the brain/placenta isoform. The PFKFB4 gene is located on chromosome 3p21–p22 and encodes the testis isozyme. 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 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 at least 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 so-called 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/dephopsphorylation 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.

Glucagon-mediated regulation of PKA activation

Representative pathway for the activation of cAMP-dependent protein kinase (PKA). In this example glucagon binds to its' cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the alpha subunit dissociates and binds to and activates adenylate cyclase. Adenylate cylcase then converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.


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 nonspherocytic hemolytic anemia.

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.

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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 tricarboxylic acid (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 glucose fermentation in cancer cells. It might seem counterintuitive that highly proliferative cells, such as is characteristic of cancers, would bypass oxidation of 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, 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.

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. 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. In contrast, PKM2 is much less active but is allosterically activated by the upstream glycolytic metabolite fructose 1,6-bisphosphate (FBP). PKM2 is also unique in that, unlike other PK isoforms, it can interact with phosphotyrosine in tyrosine phosphorylated proteins such as those resulting from growth factor stimulation of cells. The interaction of PKM2 with tyrosine phosphorylated proteins results in the release of FBP leading to reduced activity of the enzyme. Low PKM2 activity, in conjunction with increased glucose uptake, facilitates the diversion of glucose carbons into the anabolic pathways that are derived from glycolysis. 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. 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 PKM2 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.

alternate pathway of glycolysis in cancer cells

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

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.

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 supercedes 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.

Targeting PKM2 for the treatment of cancers is a distinct possibility. Recent work has demonstrated that small molecule PKM2-specific activators are functional in tumor growth models in mice. These new drugs have been shown to constitutively activate PKM2 and the activated enzyme is resistant to inhibition by tyrosine phosphorylated proteins. PKM2-specific activators reduce the incorporation of glucose into lactate and lipids. In addition, PKM2 activation results in decreased pools of nucleotide, amino acid, and lipid precursors and these effects may account for the suppression of tumorigenesis observed with these drugs.

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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.

The glucose-fatty acid cycle

The glucose-fatty acid cycle represents the interactions between glucose uptake and metabolism and the consequent inhibition of fatty acid oxidation and the effects of fatty acid oxidation on the inhibition of glucose utilization. The reciprocal regulation is most prevalent in skeletal muscle and adipose tissue. When glucose levels are high it is taken into cells via the GLUT4 transporter and phosphorylated by hexokinase. The reactions of glycolysis drive the carbon atoms to pyruvate where they are oxidized to acetyl-CoA. The fate of the acetyl-CoA is complete oxidation in the TCA cycle or return to the cytosol via citrate for conversion back to acetyl-CoA via ATP-citrate lyase (ACLY) and then into malonyl-CoA and subsequent long-chain fatty acid (LCFA) synthesis. The synthesis of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC) and once produced will inhibit the import of long-chain fatty acyl-CoAs (LCFacyl-CoA) into the mitochondria via inhibition of carnitine palmitoyltransferase 1 (CPT-1). This effectively blocks the oxidation of fatty acids leading to increased triacylglyceride synthesis (TAG). The equilibrium between malonyl-CoA synthesis and breakdown back to acetyl-CoA is determined by the regulation of ACC and malonyl-CoA decarboxylase (MCD). As long as there is sufficient capacity to divert glucose carbons to TCA cycle oxidation and fatty acid synthesis there will be limited acetyl-CoA mediated inhibition of the pyruvate dehydrogenase complex (PDHc). On the other hand, when fatty acid levels are high they enter the cell via one of several fatty acid transporter complexes [fatty acid translocase (FAT)/CD36 is shown since this transporter has a preference for LCFAs], and are then transported into the mitochondria to be oxidized. The large increase in fatty acid oxidation subsequently inhibits the utilization of glucose. This is the result of increased cytosolic citrate production from acetyl-CoA and the inhibition of phosphofructokinase-1 (PFK1). The increased acetyl-CoA derived from fat oxidation will in turn further inhibit glucose utilization via activation of PDH kinases (PDKs) that will phosphorylate and inhibit the PDHc. Although not shown, PDKs are also activated by increased mitochondrial NADH/NAD+ ratios in response to increased fatty acid β-oxidation. Under conditions where fat oxidation is favored ACC will be inhibited and MCD will be activated ensuring that LCFA that enter the cell will be able to be transported into the mitochondria. PS is pyruvate symporter responsible for mitochondrial uptake of pyruvate. TCAT is tricarboxylic acid transporter.

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 acetyl-carnitine. Mitochondrial acetyl-carnitine is formed through the action of carnitine acetyltransferase (CAT). Acetyl-carnitine is transported out of the the mitochondria via the action of carnitine-acylcarnitine translocase (CACT). Once in the cytosol acetyl-carnitine 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.

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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 re-dox 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.

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Lactate Metabolism

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 these cells is high the carbons of pyruvate will be diverted back to glucose via the gluconeogenesis pathway.

Mammalian cells contain two distinct types of LDH subunits, termed M and H. Combinations of these different subunits generates 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 the reverse.

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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.

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 signals, ACTH and growth hormone, released from the pituitary act to increase blood glucose by inhibiting uptake by extrahepatic tissues. Glucocorticoids also act to increase blood glucose levels by inhibiting glucose uptake. Cortisol, the major glucocorticoid released from the adrenal cortex, is secreted in response to the increase in circulating ACTH. The adrenal medullary hormone, epinephrine, stimulates production of glucose by activating glycogenolysis in response to stressful stimuli.

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 release. 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 muscle and brain cells 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 (and epinephrine on hepatocytes), 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 de-phosphorylation of phosphorylase thereby, inactivating it. Why is it that the glucose that enters hepatocytes is not immediately phosphorylated and oxidized? Liver cells contain an 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 freely permeable to glucose and are, therefore, essentially unaffected 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.

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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 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 membranes of kidney tubule cells and intestinal epithelial cells. There are two SGLTs in the kidney involved in glucose reabsorption. SGLT1 is found primarily in the distal S2/S3 segment of the proximal tubule and SGLT2 is expressed exclusively in the S1 segment (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.

Glucose reabsorption in kidney S1 segment of proximal tubule

Diagrammatic representation of the re-uptake of glucose in the S1 segment of the proximal tubule of the kidney by the Na+-glucose co-transporter SGLT2. Following re-uptake the glucose is transported back into the blood via the action of GLUT2 transporters. The Na+ that is reabsorbed with the glucose is transported into the blood via a (Na+-K+)-ATPase.

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 (anti-lumenal) membrane into the blood (coupled to inward uptake of potassium). 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 (and SGLT1) to reabsorb glucose is never saturated. 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 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 are currently in clinical trials with a few reaching phase III status. For information on the SGLT2 inhibitors visit the Diabetes page.

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Glucose Transporters

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.

The 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 the cell surface receptor for the human T cell leukemia virus (HTLV) is the ubiquitous GLUT1.

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Last modified: April 24, 2014