Some tissues, such as the brain, red blood cells, kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Liver glycogen, an essential postprandial source of glucose, can meet these needs for only 10–18 hours in the absence of dietary intake of carbohydrate (see Carbohydrate metabolism). During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose is formed from precursors such as lactate, pyruvate, glycerol (derived from the backbone of triacylglycerols, see Release of fatty acids from TAG), and ?-ketoacids (derived from the catabolism of glucogenic amino acids, see Release of fatty acids from TAG).
The formation of glucose does not occur by a simple reversal of glycolysis, because the overall equilibrium of glycolysis strongly favors pyruvate formation. Instead, glucose is synthesized by a special pathway, gluconeogenesis, that requires both mitochondrial and cytosolic enzymes. During an overnight fast, approximately 90% of gluconeogenesis occurs in the liver, with the kidneys providing 10% of the newly synthesized glucose molecules. However, during prolonged fasting, the kidneys become major glucose-producing organs, contributing an estimated 40% of the total glucose production. Figure 10.1 shows the relationship of gluconeogenesis to other important reactions of intermediary metabolism.
Figure 10.1.The gluconeogenesis pathway shown as part of the essential pathways of energy metabolism.
The numbered reactions are unique to gluconeogenesis. (See Figure 8.2, for a more detailed map of metabolism.)
Substrates for Gluconeogenesis
Gluconeogenic precursors are molecules that can be used to produce a net synthesis of glucose. They include intermediates of glycolysis and the tricarboxylic acid (TCA) cycle. Glycerol, lactate, and the ?-keto acids obtained from the transamination of glucogenic amino acids are the most important gluconeogenic precursors.
Glycerol is released during the hydrolysis of triacylglycerols in adipose tissue (see Fate of glycerol), and is delivered by the blood to the liver. Glycerol is phosphorylated by glycerol kinase to glycerol phosphate, which is oxidized by glycerol phosphate dehydrogenase to dihydroxyacetone phosphate—an intermediate of glycolysis. [Note: Adipocytes cannot phosphorylate glycerol because they essentially lack glycerol kinase.]
Lactate is released into the blood by exercising skeletal muscle, and by cells that lack mitochondria, such as red blood cells. In the Cori cycle, bloodborne glucose is converted by exercising muscle to lactate, which diffuses into the blood. This lactate is taken up by the liver and reconverted to glucose, which is released back into the circulation (Figure 10.2).
Figure 10.2.The Cori cycle
C. Amino acids
Amino acids derived from hydrolysis of tissue proteins are the major sources of glucose during a fast. ?-Ketoacids, such as ?-ketoglutarate, are derived from the metabolism of glucogenic amino acids (see Glucogenic and Ketogenic Amino Acids). These ?-ketoacids can enter the TCA cycle and form oxaloacetate (OAA)—a direct precursor of phosphoenolpyruvate (PEP). [Note: Acetyl coenzyme A (CoA) and compounds that give rise only to acetyl CoA (for example, acetoacetate and amino acids such as lysine and leucine) cannot give rise to a net synthesis of glucose. This is due to the irreversible nature of the pyruvate dehydrogenase reaction, which converts pyruvate to acetyl CoA (see Oxidative decarboxylation of pyruvate). These compounds give rise instead to ketone bodies (see Ketone Bodies: An Alternate Fuel for Cells) and are therefore termed ketogenic.]
Reactions Unique to Gluconeogenesis
Seven glycolytic reactions are reversible and are used in the synthesis of glucose from lactate or pyruvate. However, three of the reactions are irreversible and must be circumvented by four alternate reactions that energetically favor the synthesis of glucose. These reactions, unique to gluconeogenesis, are described below.
A. Carboxylation of pyruvate
The first “roadblock” to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of PEP to pyruvate by pyruvate kinase. In gluconeogenesis, pyruvate is first carboxylated by pyruvate carboxylase to OAA, which is then converted to PEP by the action of PEP-carboxykinase (Figure 10.3).
Figure 10.3.Activation and transfer of CO2 to pyruvate, followed by transport of oxaloacetate to the cytosol and subsequent decarboxylation.
Alternatively, OAA can be converted to PEP that is transported out of the mitochondria.
1. Biotin is a coenzyme
Pyruvate carboxylase requires biotin (see Biotin) covalently bound to the ?-amino group of a lysine residue in the enzyme (see Figure 10.3). Hydrolysis of ATP drives the formation of an enzyme–biotin–CO2 intermediate. This high-energy complex subsequently carboxylates pyruvate to form OAA. [Note: This reaction occurs in the mitochondria of liver and kidney cells, and has two purposes: to provide an important substrate for gluconeogenesis, and to provide OAA that can replenish the TCA cycle intermediates that may become depleted, depending on the synthetic needs of the cell. Muscle cells also contain pyruvate carboxylase, but use the OAA produced only for the latter purpose—they do not synthesize glucose.]
Pyruvate carboxylase is one of several carboxylases that require biotin. Others include acetyl CoA carboxylase (Carboxylation of acetyl CoA to form malonyl CoA), propionyl CoA carboxylase (Synthesis of d-methylmalonyl CoA), and methylcrotonyl CoA carboxylase (Amino acids that form acetyl CoA or acetoacetyl CoA)
2. Allosteric regulation
Pyruvate carboxylase is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA in mitochondria signal a metabolic state in which the increased synthesis of OAA is required. For example, this occurs during fasting, when OAA is used for the synthesis of glucose by gluconeogenesis in the liver and kidney. Conversely, at low levels of acetyl CoA, pyruvate carboxylase is largely inactive, and pyruvate is primarily oxidized by the pyruvate dehydrogenase complex to produce acetyl CoA that can be further oxidized by the TCA cycle (see Oxidative decarboxylation of pyruvate).
B. Transport of oxaloacetate to the cytosol
OAA must be converted to PEP for gluconeogenesis to continue. The enzyme that catalyzes this conversion is found in both the mitochondria and the cytosol in humans. The PEP that is generated in the mitochondria is transported to the cytosol by a specific transporter, whereas that generated in the cytosol requires the transport of OAA from the mitochondria to the cytosol. However, OAA is unable to directly cross the inner mitochondrial membrane; it must first be reduced to malate by mitochondrial malate dehydrogenase.
Malate can be transported from the mitochondria to the cytosol, where it is reoxidized to oxaloacetate by cytosolic malate dehydrogenase as NAD+ is reduced (see Figure 10.3). The NADH produced is used in the reduction of 1,3-BPG to glyceraldehyde 3-phosphate (see Oxidation of glyceraldehyde 3-phosphate), a step common to both glycolysis and gluconeogenesis.
C. Decarboxylation of cytosolic oxaloacetate
Oxaloacetate is decarboxylated and phosphorylated to PEP in the cytosol by PEP-carboxykinase (also referred to as PEPCK). The reaction is driven by hydrolysis of guanosine triphosphate (GTP, see Figure 10.3). The combined actions of pyruvate carboxylase and PEP-carboxykinase provide an energetically favorable pathway from pyruvate to PEP. Then, PEP is acted on by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate.
The pairing of carboxylation with decarboxylation, as seen in gluconeogenesis, drives reactions that would otherwise be energetically unfavorable. A similar strategy is used in fatty acid synthesis (see De Novo Synthesis of Fatty Acids).
D. Dephosphorylation of fructose 1,6-bisphosphate
Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase bypasses the irreversible phosphofructokinase-1 reaction, and provides an energetically favorable pathway for the formation of fructose 6-phosphate (Figure 10.4). This reaction is an important regulatory site of gluconeogenesis.
Figure 10.4.Dephosphorylation of fructose 1,6-bisphosphate.
1. Regulation by energy levels within the cell
Fructose 1,6-bisphosphatase is inhibited by elevated levels of adenosine monophosphate (AMP), which signal an “energy-poor” state in the cell. Conversely, high levels of ATP and low concentrations of AMP stimulate gluconeogenesis, an energy-requiring pathway.
2. Regulation by fructose 2,6-bisphosphate
Fructose 1,6-bisphosphatase, found in liver and kidney, is inhibited by fructose 2,6-bisphosphate, an allosteric effector whose concentration is influenced by the level of circulating glucagon (Figure 10.5). [Note: The signals that inhibit (low energy, high fructose 2,6-bisphosphate) or favor (high energy, low fructose 2,6-bisphosphate) gluconeogenesis have the opposite effect on glycolysis, providing reciprocal control of the pathways that synthesize and oxidize glucose (see During the well-fed state).]
Figure 10.5.Effect of elevated glucagon on the intracellular concentration of fructose 2,6-bisphosphate in the liver.
PFK-2 = phosphofructokinase-2; FBP-2 = fructose bisphosphatase-2.
E. Dephosphorylation of glucose 6-phosphate
Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase bypasses the irreversible hexokinase reaction, and provides an energetically favorable pathway for the formation of free glucose (Figure 10.6). Liver and kidney are the only organs that release free glucose from glucose 6-phosphate. This process actually requires two proteins: glucose 6-phosphate translocase, which transports glucose 6-phosphate across the endoplasmic reticulum (ER) membrane, and the ER enzyme, glucose 6-phosphatase (found only in gluconeogenic cells), which removes the phosphate, producing free glucose (see Figure 10.6).
[Note: These proteins are also required for the final step of glycogen degradation (see Lysosomal degradation of glycogen). Type Ia glycogen storage disease (see Figure 11.8), due to an inherited deficiency of glucose 6-phosphatase, is characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for releasing free glucose and phosphate back into the cytosol and, for glucose, into blood. [Note: Muscle lacks glucose 6-phosphatase, and therefore muscle glycogen can not be used to maintain blood glucose levels.]
Figure 10.6.Dephosphorylation of glucose 6-phosphate allows release of free glucose from liver and kidney into blood.
F. Summary of the reactions of glycolysis and gluconeogenesis
Of the 11 reactions required to convert pyruvate to free glucose, seven are catalyzed by reversible glycolytic enzymes (Figure 10.7). The irreversible reactions of glycolysis catalyzed by hexokinase, phosphofructokinase-1, and pyruvate kinase are circumvented by glucose 6-phosphatase, fructose 1,6-bisphosphatase, and pyruvate carboxylase/PEP-carboxykinase. In gluconeogenesis, the equilibria of the seven reversible reactions of glycolysis are pushed in favor of glucose synthesis as a result of the essentially irreversible formation of PEP, fructose 6-phosphate, and glucose catalyzed by the gluconeogenic enzymes. [Note: The stoichiometry of gluconeogenesis from pyruvate couples the cleavage of six high-energy phosphate bonds and the oxidation of two NADH with the formation of each molecule of glucose (see Figure 10.7).]
Figure 10.7.Summary of the reactions of glycolysis and gluconeogenesis, showing the energy requirements of gluconeogenesis.
Regulation of Gluconeogenesis
The moment-to-moment regulation of gluconeogenesis is determined primarily by the circulating level of glucagon, and by the availability of gluconeogenic substrates. In addition, slow adaptive changes in enzyme activity result from an alteration in the rate of enzyme synthesis or degradation, or both. [Note: Hormonal control of the glucoregulatory system is presented in Chapter 23.]
This hormone from the ? cells of pancreatic islets (see Glucagon) stimulates gluconeogenesis by three mechanisms.
1. Changes in allosteric effectors
Glucagon lowers the level of fructose 2,6-bisphosphate, resulting in activation of fructose 1,6-bisphosphatase and inhibition of phosphofructokinase-1, thus favoring gluconeogenesis over glycolysis (see Figure 10.5). [Note: See During the well-fed state for the role of fructose 2,6-bisphosphate in the regulation of glycolysis.]
2. Covalent modification of enzyme activity
Glucagon binds its G protein-coupled receptor (see GTP-dependent regulatory proteins) and, via an elevation in cyclic AMP (cAMP) level and cAMP-dependent protein kinase activity, stimulates the conversion of hepatic pyruvate kinase to its inactive (phosphorylated) form. This decreases the conversion of PEP to pyruvate, which has the effect of diverting PEP to the synthesis of glucose (Figure 10.8).
Figure 10.8.Covalent modification of pyruvate kinase results in inactivation of the enzyme.
OAA = oxaloacetate. [Note: Only the hepatic isozyme is subject to covalent regulation.]
3. Induction of enzyme synthesis
Glucagon increases the transcription of the gene for PEP-carboxykinase, thereby increasing the availability of this enzyme as levels of its substrate rise during fasting. [Note: Insulin causes decreased transcription of the mRNA for this enzyme.]
B. Substrate availability
The availability of gluconeogenic precursors, particularly glucogenic amino acids, significantly influences the rate of hepatic glucose synthesis. Decreased levels of insulin favor mobilization of amino acids from muscle protein, and provide the carbon skeletons for gluconeogenesis. In addition, ATP and NADH, coenzymes-cosubstrates required for gluconeogenesis, are primarily provided by the catabolism of fatty acids.
C. Allosteric activation by acetyl CoA
Allosteric activation of hepatic pyruvate carboxylase by acetyl CoA occurs during fasting. As a result of increased lipolysis in adipose tissue, the liver is flooded with fatty acids (see Increased synthesis of ketone bodies). The rate of formation of acetyl CoA by ?-oxidation of these fatty acids exceeds the capacity of the liver to oxidize it to CO2 and H2O.
As a result, acetyl CoA accumulates and leads to activation of pyruvate carboxylase. [Note: Acetyl CoA inhibits pyruvate dehydrogenase (by activating PDH kinase, see Pyruvate dehydrogenase deficiency). Thus, this single compound can divert pyruvate toward gluconeogenesis and away from the TCA cycle.]
D. Allosteric inhibition by AMP
Fructose 1,6-bisphosphatase is inhibited by AMP—a compound that activates phosphofructokinase-1. This results in a reciprocal regulation of glycolysis and gluconeogenesis seen previously with fructose 2,6-bisphosphate (see Regulation by fructose 2,6-bisphosphate). [Note: Elevated AMP thus stimulates pathways that oxidize nutrients to provide energy for the cell.]
Gluconeogenic precursors include the intermediates of glycolysis and the citric acid cycle, glycerol released during the hydrolysis of triacylglycerols in adipose tissue, lactate released into the blood by cells that lack mitochondria and by exercising skeletal muscle, and ?-ketoacids derived from the metabolism of glucogenic amino acids (Figure 10.9). Seven of the reactions of glycolysis are reversible and are used for gluconeogenesis in the liver and kidneys. Three reactions are physiologically irreversible and must be circumvented. These reactions are catalyzed by the glycolytic enzymes pyruvate kinase, phosphofructokinase, and hexokinase.
Pyruvate is converted to oxaloacetate (OAA) and then to phosphoenolpyruvate (PEP) by pyruvate carboxylase and PEP-carboxykinase. The carboxylase requires biotin and ATP, and is allosterically activated by acetyl CoA. PEP-carboxykinase requires GTP. The transcription of its mRNA is increased by glucagon and decreased by insulin. Fructose 1,6-bisphosphate is converted to fructose 6-phosphate by fructose 1,6-bisphosphatase. This enzyme is inhibited by elevated levels of AMP and activated when ATP levels are elevated.
The enzyme is also inhibited by fructose 2,6-bisphosphate, the primary allosteric activator of glycolysis. Glucose 6-phosphate is converted to glucose by glucose 6-phosphatase. This enzyme of the ER is required for the final step in gluconeogenesis, as well as hepatic and renal glycogen degradation. A deficiency of this enzyme results in severe, fasting hypoglycemia.
Figure 10.9.Key concept map for gluconeogenesis.