on 18-Mar-2018 (Sun)

Some tissues, such as the brain, red blood cells, kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continu- ous 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 p. 329). 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 p. 190), and α-ketoacids (derived from the catabolism of glucogenic amino acids, see p. 261). The formation of glucose does not occur by a simple rever- sal 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 cytoso- lic enzymes. During an overnight fast, approximately 90% of gluconeo- genesis 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 esti- mated 40% of the total glucose production. Figure 10.1 shows the rela- tionship of gluconeogenesis to other important reactions of intermediary metabolism.

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 α-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 adi- pose tissue (see p. 190), and is delivered by the blood to the liver. Glycerol is phosphorylated by glycerol kinase to glycerol phos- phate, which is oxidized by glycerol phosphate dehydrogenase to dihydroxy acetone phosphate—an intermediate of glycolysis. [Note: Adipo cytes 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).

Amino acids derived from hydrolysis of tissue proteins are the major sources of glucose during a fast. α-Ketoacids, such as α-keto - glutarate, are derived from the metabolism of glucogenic amino acids (see p. 261). These α-ketoacids can enter the TCA cycle and form oxalo acetate (OAA)—a direct precursor of phosphoenol - pyruvate (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 p. 109). These compounds give rise instead to ketone bodies (see p. 195) and are therefore termed ketogenic.

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

Carboxylation of pyruvate The first “roadblock” to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of PEP to pyru- vate by pyruvate kinase. In gluconeogenesis, pyruvate is first car- boxylated by pyruvate carboxylase to OAA, which is then converted to PEP by the action of PEP- carboxykinase

Biotin is a coenzyme: Pyruvate carboxylase requires biotin (see p. 381) covalently bound to the ε-amino group of a lysine residue in the enzyme (see Figure 10.3). Hydrolysis of ATP drives the forma- tion of an enzyme–biotin–CO 2 intermediate. This high-energy com- plex 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 gluconeo- genesis, and to provide OAA that can replenish the TCA cycle intermediates that may become depleted, depending on the syn- thetic needs of the cell. Muscle cells also contain pyruvate carboxylase , but use the OAA produced only for the latter pur- pose—they do not synthesize glucose.]

Allosteric regulation: Pyruvate carboxylase is allosterically acti- vated 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 p. 109)

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 mitochon- dria 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 dehydro genase . 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 p. 101), a step common to both glycolysis and gluconeogenesis.

Decarboxylation of cytosolic oxaloacetate Oxaloacetate is decarboxylated and phosphorylated to PEP in the cytosol by PEP-carboxykinase (also referred to as PEPCK ). The reac- tion is driven by hydrolysis of guanosine triphosphate (GTP, see Figure 10.3). The combined actions of pyruvate carboxyl ase and PEP- carboxy kinase 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 decarboxyla- tion, as seen in gluconeogenesis, drives reac- tions that would otherwise be energetically unfavorable. A similar strategy is used in fatty acid synthesis

Dephosphorylation of fructose 1,6-bisphosphate Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphos- phatase 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. 1. Regulation by energy levels within the cell: Fructose 1,6-bisphos - phatase is inhibited by elevated levels of adenosine monophos- phate (AMP), which signal an “energy-poor” state in the cell.

high levels of ATP and low concentrations of AMP stimulate gluconeogenesis, an energy-requiring pathway.

Regulation by fructose 2,6-bisphosphate: Fructose 1,6 -bis phos - phatase , 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-bis- phosphate) 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 glu- cose (see p. 100).]

Dephosphorylation of glucose 6- phosphate allows release of free glucose from liver and kidney into blood.

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) mem- brane, 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 p. 130). Type Ia glyco- gen storage disease (see p. 130), due to an inherited deficiency of glucose 6-phosphatase , is characterized by severe fasting hypo- glycemia, 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.]

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 gluco- neogenic 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).

Glucagon This hormone from the α cells of pancreatic islets (see p. 313) stim- ulates gluconeo genesis by three mechanisms. 1. Changes in allosteric effectors: Glucagon lowers the level of fruc- tose 2,6-bisphosphate, resulting in activation of fructose 1,6-bis- phosphatase and inhibition of phosphofructokinase-1, thus favoring gluconeo genesis over glycolysis

2. Covalent modification of enzyme activity: Glucagon binds its G protein-coupled receptor (see p. 95) 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).

3. Induction of enzyme synthesis: Glucagon increases the transcrip- tion 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 syn- thesis. Decreased levels of insulin favor mobilization of amino acids from muscle protein, and provide the carbon skeletons for gluconeo- genesis. 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 p. 330). The rate of formation of acetyl CoA by β-oxidation of these fatty acids exceeds the capacity of the liver to oxidize it to CO 2 and H 2 O. As a result, acetyl CoA accumulates and leads to activation of pyruvate carboxy- lase . [Note: Acetyl CoA inhibits pyruvate dehydrogenase (by activat- ing PDH kinase , see p. 111). 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 regula- tion of glycolysis and gluconeogenesis seen previously with fructose 2,6-bisphosphate (see p. 121). [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 dur- ing the hydrolysis of triacylglycerols in adipose tissue, lactate released into the blood by cells that lack mitochondria and by exercising skeletal muscle, and α-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 carboxy- lase 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 con- verted 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.

10.1 The synthesis of glucose from pyruvate by gluconeo - genesis: A. occurs exclusively in the cytosol. B. is inhibited by an elevated level of glucagon. C. requires the participation of biotin. D. involves lactate as an intermediate. E. requires the oxidation/reduction of FAD.

Which one of the following statements concerning gluco neogenesis is correct? A. It occurs in muscle. B. It is stimulated by fructose 2,6-bisphosphate. C. It is inhibited by elevated levels of acetyl CoA. D. It is important in maintaining blood glucose during the normal overnight fast. E. It uses carbon skeletons provided by degradation of fatty acids

Which one of the following reactions is unique to glu- co neo genesis? A. Lactate → pyruvate B. Phosphoenolpyruvate → pyruvate C. Oxaloacetate → phosphoenolpyruvate D. Glucose 6-phosphate → fructose 6-phosphate E. 1,3-Bis-phosphoglycerate → 3-phosphoglycerate

The metabolism of ethanol by alcohol dehydroge- nase (ADH) produces NADH. What effect is the change in the NAD + /NADH expected to have on glu- coneogenesis? Explain.

Given that acetyl CoA cannot be a substrate for glu- coneogenesis, why is its production in fatty acid oxi- dation essential for gluconeogenesis?

What effect does AMP have on gluconeogenesis and glycolysis? What enzymes are affected?