GLUCONEOGENESIS Synthesis of glucose from noncarbohydrate precursors Learning objectives: List gluconeogenic precursors List the enzymes and intermediates involved in gluconeogenesis List the irreversible and regulated steps of gluconeogenesis Discuss regulation of gluconeogenesis

Gluconeogenic precursors 18 amino acids (diet and degradation of protein) Lactate (anaerobic glycolysis) Glycerol (hydrolysis of triacylglycerols) Notable exception: Fatty acids

Gluconeogenic precursors Lactate and some amino acids can be converted to pyruvate Some amino acids can be converted to oxaloacetate Glycerol can be converted to dihydroxyacetone phosphate

Gluconeogenic pathway describes conversion of pyruvate to glucose Main organs producing glucose via the gluconeogenic pathway are liver and kidney

Gluconeogenic pathway is NOT a simple reversal of glycolysis The 3 irreversible steps of glycolysis Hexokinase/Glucokinase Phosphofructokinase Pyruvate kinase must be circumvented The 7 reversible steps of glycolysis are part of gluconeogenesis

All the intermediates of glycolysis are part of gluconeogenesis In addition, gluconeogenesis involves oxaloacetate and (indirectly) malate O O- C C O CH2 O O- C H C OH CH2 Oxaloacate Malate

Stoichiometry 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 6 H2O → 1 Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+

This reaction occurs in the mitochondria It is a regulated step Pyruvate carboxylase Pyruvate + CO2 + ATP + H2O Oxaloacetate + ADP + Pi + 2 H+ This reaction occurs in the mitochondria It is a regulated step Biotin is a coenzyme for the reaction

Pyruvate is transported from the cytoplasm to the mitochondria. In the mitochondria, pyruvate is converted to oxaloacetate by pyruvate carboxylase Oxaloacetate can not be transported to the cytoplasm. Oxaloacetate is reduced in the mitochondria to malate: Malate dehydrogenase Oxaloacetate + NADH + H+ Malate + NAD+ Malate is transported to the cytoplasm and reoxidized back to oxaloacetate: Malate + NAD+ Oxaloacetate + NADH + H+

It is a regulated step Phosphoenolpyruvate carboxykinase (PEPCK) Oxaloacetate + GTP Phosphoenolpyruvate + GDP + CO2 It is a regulated step

Irreversible Regulated step Fructose-1,6- bisphosphatase Fructose 1,6-bisphosphate + H2O Fructose 6-phosphate + Pi Irreversible Regulated step

Only present in large amount in liver and kidney Glucose-6-phosphatase Glucose 6-phosphate + H2O Glucose + Pi Irreversible Regulated step Only present in large amount in liver and kidney Reaction occurs in the endoplasmic reticulum

Glycerol kinase Glycerol + ATP Glycerol phosphate + ADP + H+ Glycerol phosphate dehydrogenase Glycerol phosphate + NAD+ Dihydroxyacetone phosphate + NADH + H+

LIVER IN THE FASTED STATE Pyruvate carboxylase Pyruvate + CO2 + ATP + H2O Oxaloacetate + ADP + Pi + 2 H+ + Acetyl CoA (High energy signal) LIVER IN THE FASTED STATE Energy is derived mostly from fatty acids Pyruvate dehydrogenase Acetyl-CoA Fatty acids - Pyruvate + Oxaloacetate Pyruvate carboxylase

+ - Phosphoenolpyruvate carboxykinase (PEPCK) Oxaloacetate + GTP Phosphoenolpyruvate + GDP + CO2 Phosphoenolpyruvate carboxykinase is regulated at the level of gene transcription + Glucagon, glucocorticoids (fasted state) Insulin (fed state) -

- - + Fructose-1,6- bisphosphatase Fructose 1,6-bisphosphate + H2O Fructose 6-phosphate + Pi - AMP (low-energy state) Fructose 2,6-bisphosphate (fed state, high insulin/glucagon ratio) ATP (high-energy state) - +

+ - + Glucose-6-phosphatase Glucose 6-phosphate + H2O Glucose + Pi The catalytic subunit of glucose-6-phosphatase is regulated at the level of gene transcription + Glucagon, glucocorticoids (fasted state) Insulin (fed state) Glucose (fed state) - “Paradoxical regulation” - +

Glycolysis Gluconeogenesis Glucokinase Glucose Insulin Glucose Glucose-6-phosphatase - Insulin Glucagon Glucose + + + Glucose-6-phosphate +

Fructose 1,6-bisphosphate Glycolysis Gluconeogenesis Phosphofructokinase Fructose 6-phosphate Fructose-1,6- bisphosphatase - ATP Citrate H+ Fructose 2,6-bisphosphate AMP + ATP Fructose 2,6-bisphosphate AMP - - Fructose 1,6-bisphosphate + - + -

Glycolysis Gluconeogenesis Pyruvate kinase PEPCK Phosphoenolpyruate - Glucagon ATP Alanine Fructose 1,6-bisphosphate Glucose + Glucagon Insulin Acetyl-CoA - - Pyruvate carboxylase - + + + Pyruvate

Allosteric regulator of glycolysis and gluconeogenesis Fructose 2,6-bisphosphate Phosphofructokinase 2 Fructose 6-phosphate + ATP Fructose 2,6-bisphosphate + ADP Fructose bisphosphatase 2 Fructose 2,6-bisphosphate + H2O Fructose 6-phosphate + Pi

G-6-P F-6-P PFK 2 FBPase 2 F-2,6-P2 + - PFK 1 FBPase 1 F -1,6-P 2 + PK The PFK2 and FBPase 2 activities are located in a single protein: The bifunctional enzyme

Fasted state: High Glucagon -> High cAMP -> activation of PKA -> phosphorylation of bifunctional enzyme -> inhibition of PFK2, activation of FBPase2 -> decrease in fructose 2,6-bisphosphate -> no stimulation of glycolysis, no inhibition of gluconeogenesis -> Gluconeogenesis prevails! Fed state: Low Glucagon -> No/Low cAMP -> no activation of PKA -> dephosphorylation of bifunctional enzyme prevails -> activation of PFK2, inhibition of FBPase2 -> increase in fructose 2,6-bisphosphate -> stimulation of glycolysis, inhibition of gluconeogenesis -> Glycolysis prevails!

The Cori cycle Blood- stream LIVER MUSCLE Glucose Glucose Lactate Glucose Lactate Gluconeogenesis Glycolysis