Gluconeogenesis: Making New Glucose

• Gluconeogenesis means "making new glucose"

What is Gluconeogenesis?

• Cells depend on glucose for energy
• The brain and red blood cells depend almost entirely on glucose
• Liver-stored glycogen provides enough glucose to supply the brain for 1½ days during fasting
• If glucose isn't from diet, the body will produce glucose from non-carbohydrate precursors
• Gluconeogenesis is how new sugar is generated

Gluconeogenesis Substrates

• Lactate and pyruvate are converted into new glucose
• Most amino acids from protein hydrolysis become glucose
• Glycerol (TAG hydrolysis) forms glucose, but not fatty acids
• Animals cannot use acetyl CoA to synthesize glucose
• Plants use acetyl CoA as a substrate to make glucose via the glyoxalate cycle
• This process occurs primarily in the liver (90%) and kidneys (10%)
• Microorganisms use acetate & propionate as gluconeogenesis substrates

Gluconeogenesis and Glycolysis

• Gluconeogenesis and glycolysis are opposing metabolic pathways
• They differ in three ways, because the strongly exergonic reactions of glycolysis are bypassed:
• Pyruvate kinase is bypassed by pyruvate carboxylase & PEP carboxykinase
• Phosphofructokinase is bypassed by fructose-1,6-bisphosphatase
• Hexokinase is bypassed by glucose-6-phosphatase

Glycolysis Net Reaction

• Glucose + 2 NAD+ + 2 ADP + 2 Pi yields 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O

Gluconeogenesis Net Reaction

• 2 Pyruvate + 2 NADH + 2 H+ + 4 ATP + 2 GTP + 6 H2O yields Glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
• 2 ATP + 2 GTP + 4 H2O yields 2 ADP + 2 GDP + 4 Pi
• This is a wasteful futile cycle, so it is tightly regulated

Gluconeogenesis Enzymes

• Gluconeogenesis enzymes are in the cytosol, except:
  • Glucose-6-phosphatase in the ER
  • Pyruvate carboxylase in the mitochondria

Unique Reactions of Gluconeogenesis: Bypass I

• Pyruvate becomes oxaloacetate, which becomes phosphoenolpyruvate
• This bypasses Pyruvate kinase

Pyruvate Carboxylase

• Pyruvate carboxylase is used for carboxylation in the mitochondrial matrix
• Acetyl CoA activates this
• Bicarbonate is needed
• Biotin is a cofactor
• Oxaloacetate is too polar to pass through mitochondrial membranes, so it converts to aspartate or malate for transport

Phosphoenolpyruvate Carboxykinase

• Phosphoenolpyruvate carboxykinase facilitates decarboxylation
• It occurs in the cytosol
• Oxaloacetate is a high-energy intermediate
• Its exergonic decarboxylation provides the free energy to form PEP
• Beta-ketoacids are 'high energy' intermediates
• Loss of CO2 generates a strong enolate

Pyruvate Carboxylase details

• It is a Biotin-Dependent Enzyme
• The enzyme covalently links biotin to an active-site lysine
• The nitrogen at the bottom of the structure transfers the one-carbon unit to pyruvate
• Bicarbonate must be activated for attack by the pyruvate carbanion, which is driven by ATP

Unique Reactions of Gluconeogenesis: Bypass II

• Fructose 1,6-Bisphosphate becomes Fructose 6-Phosphate
• Phosphofructokinase is replaced

Fructose 1,6-Bisphosphatase

• It is used for Phosphoester hydrolysis
• It occurs in the cytosol

Unique Reactions of Gluconeogenesis: Bypass III

• Glucose 6-Phosphate becomes Glucose
• Hexokinase is replaced

Glucose 6-Phosphatase

• It is used for Phosphoester hydrolysis
• It occurs in the ER

Location Specificity

• Glucose 6-phosphatase is not present in the muscle or brain
• Gluconeogenesis cannot occur in these organs because of this
• Glucose is exported from the ER into the blood stream
• It goes to the brain and carries out glycolysis there

Glycogen Storage

• Higher animals store excess glucose as glycogen
• Glycogen phosphorylase facilitates Glycogen breakdown into Glucose
• Glycogen synthase facilitates the synthesis of Glu into Glycogen

Glucose Activation

• Glucose is activated by the formation of nucleotide sugars
• Sugar Nucleotides are activated sugar units
• UDP-glucose is a donor for glycogen biosynthesis in animals
• Nucleotide labels mark sugar for biosynthesis
• ADP-glucose is a donor for starch biosynthesis in plants
• UDP-glucose Pyrophosphorylase is used

Gluconeogenesis Regulation

• Glycolysis is ON when the cell needs ATP & NADH
• Gluconeogenesis is ON when the cell doesn’t need ATP & NADH and has excess lactate/pyruvate and the cell wants to store glucose as glycogen

Regulation Mechanisms

• Substrate-level control of Gluocose-6-phosphatase
• Allosteric regulation via Fructose-2,6,-Bisphosphate & Acetyl CoA
• Covalent modification (Phosphorylation/Dephosphorylation via cAMP) allows for hormonal control
• Transcriptional control of gene encoding PEP carboxykinase

Regulating Cellular processes

• Glycogen metabolism and blood glucose levels are hormonally regulated
• Insulin lowers blood glucose
• Glucagon raises blood glucose
• The body maintains blood glucose levels at approximately 4.5 mM

Insulin

• Insulin increases glucose permeability in muscle and adipose tissue, triggering glycolysis, glycogen synthesis, and triacylglycerol synthesis

Glucagon

• Glucagon increases cyclic AMP in the liver and adipose tissue, leading to glycogen breakdown and high blood glucose levels

Epinephrine

• Epinephrine increases cyclic AMP levels in skeletal muscle, leading to triacylglycerol hydrolysis, glycogen breakdown, and increased blood glucose levels

Cortisol and Glucocorticoid

• Cortisol and Glucocorticoid are hormones used for long-term stress
• Glucocorticoid affects the liver, skeletal muscle, and adipose tissue
• It promotes protein breakdown and decreases protein synthesis
• It stimulates gluconeogenesis and glycogen synthesis (transcriptional control)

Glucose Metabolism Reactions

• The pentose phosphate pathway operates in the liver & adipose tissue & generates NADPH (reducing power) & ribose-5-phosphate
• ATP is the cell's "energy" currency
• NADPH drives endergonic reductive biosynthesis
• The pentose phosphate pathway is an alternative pathway to glycolysis

Glucose Metabolism Overall Net Reaction

• 3 Glucose-6-P + 6 NADP+ + 3 H2O yields 6 NADPH + 6 H+ + 3 CO2 + 2 Fructose-6-P + Glyceraldehyde-3-P

Glucose Metabolism Three Stages of Pentose Phosphate Pathway

• 1. Oxidative Reactions
• 2. Isomerization & Epimerization Reactions
• 3. C–C Bond Cleavage & Formation Reactions

Glucose-6-Phosphate

• Glucose-6-Phosphate is a crucial branch point in glycolysis & the pentose phosphate pathway

NADPH

• Flux through the pentose phosphate pathway and the rate of NADPH production are controlled at the 1st step (glucose-6-phosphate dehydrogenase)
• ATP is made by having glucose-6-P enter glycolysis
• NADPH or Ribose-5-P are made by having glucose-6-P enter the pentose phosphate pathway

Possibilities for Pentose Phosphate Pathway

• (1) Both ribose-5-phosphate & NADPH are needed
• (2) More ribose-5-phosphate than NADPH is needed
• (3) More NADPH than ribose-5-phosphate is needed
• (4) Both NADPH & ATP are needed, but ribose-5-phosphate is not