UNIT II: Intermediary Metabolism Gluconeogenesis

Figure 10.1. The gluconeogenesis pathway shown as part of the essential pathways of energy metabolism. The numbered reactions are unique to gluconeogenesis..

Overview Some tissues e.g., brain, RBCs, kidney medulla, lens & cornea, testes, & exercising muscle, require a continuous supply of gluc as a metabolic fuel. Liver glycogen, an essential postprandial source of gluc, can meet these needs for only 10-18 h in the absence of dietary intake of CHO During a prolonged fast, hepatic glycogen stores are depleted, & gluc is formed from precursors such as lactate, pyruvate, glycerol (from backbone of triglycerols), and α-ketoacids (from catabolism of glucogenic aa’s) The formation of gluc does not occur by simple reversal of glycolysis, because overall equil of glycolysis strongly favors pyruvate formation Instead gluc is synthesized by a special pathway, gluconeogenesis During an o/n fast, ~ 90% of gluconeogenesis occurs in liver, with kidneys providing 10% of the newly synthesized gluc molecules However, during prolonged fasting, kidneys become major gluc-producing organs, contributing an estimated 40% of the total gluc production

II. Substrates for gluconeogenesis Gluconeogenic precursors are molecules that can be used to produce a net synthesis of gluc. They include all the intermediates of glycolysis and TCA cycle. Glycerol, lactate, and α-keto acids obtained from deamination of glucogenic aa’s are the most important gluconeogenic precursors A. Glycerol Is released during the hydrolysis of triglycerols in adipose tissue, & is delivered by blood to the liver Glycerol is phosphorylated by glycerol kinase to glycerol-P, which is oxidized by glycerol phosphate dehydrogenase to DHAP = an intermediate of glycolysis Note: adipocytes can’t phosphorylate glycerol because they lack glycerol kinase

B. Lactate Lactate is released into blood by exercising skeletal muscle, and by cells that lack mitoch e.g., RBCs In the Cori cycle, blood-borne gluc is converted by exercising muscle to lactate, which diffuses into the blood. This lactate is taken up by the liver and converted to gluc, which is released back into circulation Figure 10.2. The Cori cycle.

C. Amino acids Amino acids derived from hydrolysis of tissue proteins are the major sources of gluc during a fast. α-ketoacids, e.g., OAA and α-KG, are derived from the metabolism of glucogenic amino acids. These substances can enter the TCA cycle and form OAA, a direct precursor of PEP Note: Acetyl CoA & cpds that give rise to acetyl CoA (e.g., acetoacetate and aa’s such as Lys and Leu) can’t give rise to a net synthesis of gluc. This is due to the irreversible nature of the pyruvate dehydrogenase reaction, which converts pyruvate to acetyl CoA. These cpds give rise to ketone bodies and are therefore temred ketogenic

III. Reactions unique to gluconeogenesis Seven glycolytic reactions are reversible and are used in the synthesis of gluc from pyruvate or lactate. However, 3 of the reactions are irreversible and must be circumvented by 4 alternate reactions that energetically favor synthesis of gluc A. Carboxylation of pyruvate The 1st “roadblock” to overcome in synthesis of gluc from pyruvate is the irreversible conversion in glycolysis of pyruvate to PEP by pyruvate kinase In gluconeogenesis pyruvate is 1st carboxylated by pyruvate carboxylase to OAA, which is then converted to PEP by PEP-carboxykinase

Figure 10.3. Activation and transfer of CO2 to pyruvate, followed by transport of oxaloacetate to the cytosol and subsequent decarboxylation.

1. Biotin is a coenzyme: Pyruvate carboxylase contains biotin, which is covalently bound to the enz protein through the ε-amino group of Lys, forming an active enz This covalently bound form of biotin is called biocytin Cleavage of a high-energy phosphate of ATP drives formation of an enz-biotin-CO2 intermediate. This high-energy complex subsequently carboxylates pyruvate to form OAA Note: this reaction occurs in the mitoch of liver & kidney cells, & has 2 purposes: to provide an important substrate for gluconeogenesis, & to provide OAA that can replenish TCA cycle intermediates that may become depleted, depending on synthetic needs of the cell. Muscle cells also contain pyruvate carboxylase, but use OAA produced only for the latter purpose, they do not synthesize glucose.

2. Allosteric regulation: - Pyruvate carboxylase is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA may signal one of several metabolic states in which the increased synthesis of OAA is required. - e.g., this may occur during fasting in which OAA is used for synthesis of gluc by gluconeogenesis in the liver & kidney. - Conversely, at low levels of acetyl CoA, pyruvate carboxylase is largely inactive, & pyruvate is primarily oxidized by pyruvate dehydrogenase to produce acetyl CoA that can be further oxidized by the TCA cycle.

B. Transport of OAA to the cytosol OAA, formed in mitoch., must enter cytosol where the other enz’s of gluconeogenesis are located. However, OAA is unable to directly cross inner mitoch memb; it must first be reduced to malate by mitoch malate dehydrogenase Malate can be transported from mitoch to cytosol, where it is reoxidized to OAA by cytosol malate dehydrogenase C. Decarboxylation of cytoslic OAA OAA is decarboxylated & phosphorylated in cytosol by PEP-carboxykinase (= PEPCK). The reaction is driven by hydrolysis of GTP. The combined actions of pyruvate carboxylase & PEPCK provide an energetically favorable pathway from pyruvate to PEP. PEP is then acted on by the reactions of glycolysis running in the reverse direction until it becomes F-1,6-BP

D. Dephosphorylation of fructose 1,6-bisphosphate Hydrolysis of F-1,6-BP by fructose 1,6-bisphosphatase bypasses the irreversible PFK-1 reaction, & provides an energetically favorable pathway for formation of F-6-P. This reaction is an important regulatory site of gluconeogenesis 1. Regulation by energy levels within the cell: - fructose 1,6-bisphosphatase is inhibited by elevated levels of AMP, which signal an “energy-poor” state in the cell. Conversely, high levels of ATP & low conc’s of AMP stimulate gluconeogenesis 2. Regulation by fructose 2,6-bisphosphate: - Fructose 1,6-bisphosphatase, found in liver & kidney, is inhibited by fructose 2,6-bisphosphate, an allosteric modifier whose conc is influenced by the level of circulating glucagon Note: recall that F-2,6-BP activates PFK-1 of glycolysis, thus allowing for reciprocal control of gluc synthesis & oxidation

Figure 10.4 Dephosphorylation of fructose 1,6-bisphosphate.

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 bisphospate phosphatase-2.

E. Dephosphorylation of glucose 6-phosphate Hydrolysis of G-6-P by glucose 6-phosphatase bypasses the irreversible hexokinase reaction, & provides an energetically favorable pathway for the formation of free gluc. Liver & kidney are the only organs that release free gluc from G-6-P This process actually requires 2 enz’s: glucos 6-phosphate translocase, which transports G-6-P across the ER memb, & a 2nd ER enz, glucose 6-phosphatase (found only in gluconeogenic cells), which removes P, producing free gluc.

Figure 10.6. Dephosphorylation of glucose 6-phosphate.

Note: these enz’s are required for the final steps in glycogenolysis, as well as gluconeogenesis - Type Ia glycogen storage disease (Von Gierke disease) results from an inherited deficiency of one of these enzymes - Specific transporters are responsible for releasing free gluc & P back into cytosol, & in hepatocytes, into the blood Note: muscle lacks glucose 6-phosphatase & therefore, can’t provide blood gluc by gluconeogenesis. Also, G-6-P derived from muscle glycogen can’t be dephosphorylated to yield free gluc.

F. Summary of the reactions of glycolysis & gluconeogenesis Of the 11 reactions required to convert pyruvate to free gluc, 7 are catalyzed by reversible glycolytic enz’s. The irreversible reactions of glycolysis catalyzed by hexokinase, PFK, & pyruvate kinase are circumvented by glucose 6-phosphatase, fructose 1,6-bisphosphatase & pyruvate carboxylase/PEP carboxykinase In gluconeogenesis, the equilibria of the 7 reactions of glycolysis are pushed in favor of gluc synthesis as a result of the essentially irreversible formation of PEP, F-6-P, & glucose catalyzed by gluconeogenic enz’s. Note: stoichiometry of gluconeogenesis from pyruvate couples cleavage of 6 high-energy phosphate bonds & oxidation of 2 NADH with formation of each molecule of glucose

Figure 10.7. Summary of the reactions of glycolysis and gluconeogenesis, showing the energy requirements of gluconeogenesis.

IV. Regulation of gluconeogenesis The moment-to-moment regulation of gluconeogenesis is determined primarily by the circulating level of glucagon, and by availability of gluconeogenic substrates In addition, slow adaptive changes in enz activity result from an alteration in rate of enz synthesis or degradation, or both A. Glucagon This pancreatic islet hormone stimulates gluconeogenesis by 3 mechanisms 1. Changes in allosteric effectors: - Glucagon lowers level of fructose 2,6-BP, resulting in activation of fructose 1,6-bisphosphatase & inhibition of PFK1

2. Covalent modification of enzyme activity: Glucagon, via an elevation of cAMP level & cAMP-dependent protein kinase activity, stimulates the conversion of pyruvate kinase to its inactive (phosphorylated) form This decreases conversion of PEP to pyruvate, which has the effect of diverting PEP to the synthesis of glucose 3. Induction of enzyme synthesis: Glucagon increases transcription of PEP carboxykinase gene, thereby increasing the availability of this enzyme’s activity as levels of its substrate rise during fasting Note: insulin causes decreased transcription of mRNA for this enz

Figure 10.8 Covalent modification of pyruvate kinase results in inactivation of the enzyme. OAA = oxaloacetate.

B. Substrate availability Availability of gluconeogenic precursors, particularly glucogenic aa’s, significantly influences the rate of hepatic glucose synthesis Decreased levels of insulin favor mobilization of aa’s from muscle protein, & provide the C skeletons for gluconeogenesis C. Allosteric activation by acetyl CoA Allosteric activation of hepatic pyruvate carboxylase by acetyl CoA occurs during fasting As a result of excessive lipolysis in adipose tissue, the liver is flooded with fatty acids. The rate of formation of acetyl CoA by β-oxidation of these fatty acids exceeds capacity of liver to oxidize it to CO2 & H2O As a result, acetyl CoA accumulates and leads to activation of pyruvate carboxylase Note: acetyl CoA inhibits pyruvate dehydrogenase . Thus, this single cpd can divert pyruvate toward gluconeogenesis & away from TCA cycle

D. Allosteric inhibition by AMP Fructose 1,6 bisphosphatase is inhibited by AMP, a cpd that activates PFK1 Elevated AMP thus stimulates pathways that oxidize nutrients to provide energy for the cell Note: ATP & NADH, produced in large quantities during fasts by catalytic pathways, such as fatty acid oxidation, are required for gluconeogenesis

Summary Gluconeogenic precursors include all intermediates of glycolysis & TCA cycle, glycerol released during hydrolysis of triglycerols in adipose tissue, lactate released into blood by cells that lack mitoch & by exercising skeletal muscle, & α-ketoacids derived from metabolism of glucogenic aa’s. Seven of glycolysis reactions are reversible & are used for gluoneogenesis in the liver & kidneys. Three reactions are physiologically irreversible & must be circumvented. These reactions are catalyzed by glycolytic enz’s pyruvate kinase, PFK, & hexokinase Pyruvate is converted to PEP by pyruvate carboxylase & PEP carboxykinase the carboxylase requires biotin & ATP, & is allosterically activated by acetyl CoA

PEP carboxykinase requires GTP PEP carboxykinase requires GTP. The transcription of its mRNA is increased by glucagon & decreased by insulin F 1,6-bisphosphate is converted to F 1-P by F 1,6-bisphosphatase. This enz is inhibited by elevated levels of AMP & activated by elevated levels of ATP. The enz is also inhibited by F 2,6-bisphosphate, the primary allosteric activator of glycolysis. G-6-P is converted to gluc by G-6-phosphatase. This enz activity is required for the final step in glycogen degradation, as well as gluconeogenesis. A deficiency of this enz results in type Ia glycogen storage disease

Figure 10.9 Key concept map for gluconeogenesis.