GLUCONEOGENESIS Synthesis of glucose from noncarbohydrate precursors Learning

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 OC OC C O O O H C OH CH 2 C C O- Oxaloacate O O- Malate

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

Pyruvate carboxylase Pyruvate + CO 2 + ATP + H 2 O 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: Oxaloacetate + NADH + H+ Malate dehydrogenase Malate + NAD+ Malate is transported to the cytoplasm and reoxidized back to oxaloacetate: Malate dehydrogenase Malate + NAD+ Oxaloacetate + NADH + H+

Phosphoenolpyruvate carboxykinase (PEPCK) Oxaloacetate + GTP It is a regulated step Phosphoenolpyruvate + GDP + CO 2

Fructose-1, 6 bisphosphatase Fructose 1, 6 -bisphosphate + H 2 O Irreversible Regulated step Fructose 6 -phosphate + P i

Glucose-6 -phosphatase Glucose 6 -phosphate + H 2 O Glucose + P i 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+

Pyruvate carboxylase Pyruvate + CO 2 + ATP + H 2 O Oxaloacetate + ADP + Pi + 2 H+ + Acetyl Co. A (High energy signal) LIVER IN THE FASTED STATE Energy is derived mostly from fatty acids Pyruvate dehydrogenase Acetyl-Co. A Pyruvate carboxylase Fatty acids + Oxaloacetate

Phosphoenolpyruvate carboxykinase (PEPCK) Oxaloacetate + GTP Phosphoenolpyruvate + GDP + CO 2 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 + H 2 O + Fructose 6 -phosphate + P i AMP (low-energy state) Fructose 2, 6 -bisphosphate (fed state, high insulin/glucagon ratio) ATP (high-energy state)

Glucose-6 -phosphatase Glucose 6 -phosphate + H 2 O Glucose + P i 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 Glucokinase + + Gluconeogenesis Glucose-6 -phosphatase Insulin Glucose-6 -phosphate + + Insulin Glucagon Glucose

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

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

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 + H 2 O Fructose 6 -phosphate + Pi

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

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

The Cori cycle LIVER Glucose Gluconeogenesis Lactate Bloodstream MUSCLE Glucose Glycolysis Lactate