Gluconeogenesis: Glucose Synthesis

• Gluconeogenesis refers to making new glucose

Energy and Glucose Dependence

• Cells depend on glucose for energy
• Brain and red blood cells are almost entirely dependent on glucose for energy
• Liver glycogen stores are only sufficient to supply the brain with glucose for 1/2 a day when fasting
• If glucose is not obtained from diet, the body produces it from non-carbohydrate precursors

Gluconeogenesis Overview

• Gluconeogenesis defines the generation of new sugar
• Lactate and pyruvate are converted to glucose through the Cori Cycle
• Most amino acids from protein hydrolysis act as protein sources
• Glycerol from TAG hydrolysis, but not fatty acids is used to synthesize glucose
• Animals cannot use acetyl CoA to produce glucose
• Plants use acetyl CoA as a substrate through the glyoxalate cycle
• Gluconeogenesis takes place in the liver (90%) and kidney (10%)
• Microorganisms utilize acetate and propionate as substrates

Relationship to Glycolysis

• Gluconeogenesis and glycolysis are opposing metabolic pathways
• Three reactions are bypassed:
  • Pyruvate kinase is bypassed by pyruvate carboxylase and 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
• Add Both Net Reactions Together: 2 ATP + 2 GTP + 4 H2O à 2 ADP + 2 GDP + 4 Pi
• It is a tightly regulated wasteful futile cycle
• Gluconeogenesis enzymes occur in the cytosol with the exception of glucose-6-phosphatase, which occurs in the ER, and pyruvate carboxylase, which occurs in the mitochondria

Unique Reactions of Gluconeogenesis

• There is a bypass, pyruvate is converted to oxaloacetate, which is then converted to phosphoenolpyruvate
• Pyruvate kinase is replaced

Pyruvate Carboxylase

• Pyruvate carboxylase facilitates carboxylation in the mitochondrial matrix
• Pyruvate with just CO2 dissolved in H2O is converted with ATP to oxaloacetate with ADP + P
• Acetyl CoA activates reaction
• Biotin is a cofactor
• Oxaloacetate cannot pass through mitochondrial membranes
• Oxaloacetate is converted to aspartate or malate for transport

Phosphoenolpyruvate Carboxykinase

• PEP carboxykinase facilitates decarboxylation
• Oxaloacetate with GTP converts to PEP with GDP + CO2 and occurs in the cytosol
• Oxaloacetate is a high energy intermediate
• Exergonic decarboxylation provides the free energy to form PEP
• Beta-ketoacids are high energy intermediates because the loss of CO2 generates a strong enolate

Biotin-Dependent Enzyme

• Pyruvate carboxylase is a biotin-dependent enzyme
• There is a covalent linkage of biotin to an active-site lysine in pyruvate carboxylase
• 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
• This activation is driven by ATP

Fructose Reactions

• Fructose 1,6-bisphosphate becomes Fructose 6-Phosphate
• Phosphofructokinase is replaced
• Fructose 1,6-bisphosphatase enables phosphoester hydrolysis and always happens in the cytosol

Glucose Reactions

• Glucose 6-Phosphate becomes Glucose
• Hexokinase is replaced
• Glucose 6-Phosphatase enables phosphoester hydrolysis and always happens in the ER
• The enzyme is not found in muscle or brain, so gluconeogenesis cannot occur in these organs
• Glucose is exported from the ER into bloodstream to go to the brain and carries out glycolysis there

Regulation of Gluconeogenesis

• Glycolysis turns on when cells need ATP and NADH
• Gluconeogenesis turns on when cells not need ATP and NADH and excess lactate/pyruvate and cells storing glucose as glycogen

Mechanisms of Regulation

• Substrate-level control of Gluocose-6-phosphatase
• Allosteric regulation via Fructose-2,6,-Bisphosphate and Acetyl CoA
• Covalent modification such as Phosphorylation/Dephosphorylation via cAMP allows for hormonal control
• Transcriptional control of gene encoding PEP carboxykinase
• Inhibits Gluconeogenensis and phosphorylates and inhibits fructose-6-phosphate
• Stimulates gylcolysis and is phosphorylated and stimulated by fructose 2,6-BP creating a bifunctional enzyme

Glycogen

• Glycogen is found primarily in liver and muscle
• The structure is optimized for the ability to store and deliver energy quickly and for the longest amount of time
• A key to this optimization is the average chain length of the branches (13 residues)

Glycogen and Starch Catabolism

• Alpha-Amylase is an endoglycosidase alpha(1->4)
• It is active on either side of a branch point, but activity is reduced near the branch points
• Debranching enzyme cleaves limit dextrins
• There are 2 activities of the debranching enzyme:
  • It transfers trisaccharide groups
  • Cleaves the remaining single glucose units from the main chain alpha(1->6)

Glycogen Regulation

• Digestive breakdown of starch is unregulated as nearly 100% of ingested food is absorbed and metabolized
• Tissue glycogen is an important energy reservoir and its breakdown is carefully controlled
• Glycogen consists of granules of high MW
• Glycogen phosphorylase cleaves glucose from the nonreducing ends of glycogen molecules
• This is a phosphorolysis and not a hydrolysis
• There is a metabolic advantage as the product is a sugar-P that is a potential glycolysis substrate

Glycogen Catabolism

• Glycogen Phosphorylase is the key enzyme in liberating glucose units from stored glycogen reserves in muscles and liver when energy is needed It's tightly regulated through allosteric regulation and covalent modification
• Paradigm of enzyme regulation
• It is activated when the cell needs energy and inactivated when cells at rest, storing glucose as glycogen

Catalytic Reaction

• Cleaves glucose units from non reducing ends of glycogen
• Phosphorolysis instead of hydrolysis because Glucose-1-phosphate is the product
• G1P à G6P yields Glycolysis through phosphoglucomutase

Glycogen Phosphorylase Regulation

• It meets normal metabolic needs under normal conditions
• Positive homotropic effector stabilizes the R-state: Pi
• Negative heterotropic effectors stabilize the T-state: ATP & glucose-6-P
• Positive heterotropic effector stabilizes the R-state: AMP

Covalent Modification

• It is an emergency sitation. In fight or flight situations, covalent modification overrides everything
• Phosphorylation of Ser-14 increases the actiive phosphorylase a form that is unresponsive to allosteric regulation
• Hormone-activated signal cascade through glucagon and epinephrine

Glycogen Synthesis

• Higher animals store excess glucose as glycogen
• Glycogen phosphorylase converts Glycogen to Glucose whereas glycogen synthase converts Glucose to Glycogen
• There is an activation of glucose by formation of nucleotide sugars
• Sugar Nucleotides are activated forms of sugar units
• UDP-glucose acts as the donor for glycogen biosynthesis in animals
• Nucleotide labels mark sugar for biosynthesis
• ADP-glucose = donor for starch biosynthesis in plants
• UDP-glucose Pyrophosphorylase with Glu-1-Phosphate + UTP yields UDP-Glucose + PPi
• The reaction is driven by PPi hydrolysis
• Only one phosphoanhydride bond is consumed in the storage of one glucose in glycogen, but upon glycogen degradation, one glucose yields 32 ATP

Glycogen Synthase

• Synthesis of glycogen occurs through glycogen synthase and branching enzyme
• There is a process where UDP-Glucose + (Glucose)n yields UDP + (Glucose)n+1
• Glucose is added to non-reducing ends of glycogen by glyogen synthase
• Glycogenin (a protein) is covalently linked to the first glucose through an acetal linkage to Tyr-OH
• Alpha, a (1->6) branches every 8-12 residues that are added generates more non reducing ends

Glycogen Metalbolism Control

Hormones regulate glycogen metabolism and blood glucose levels

• Insulin lowers blood glucose, while glucagon increases it to maintain blood glucose levels
• Glycogen is metabolized by glucagon and epinephrine and insulin facilitates glycogen biosynthesis
• Glucocorticoid affects liver, Skeletal muscle, and adipose tissue, promotes protein breakdown, decreases protein synthesis, and stimulates gluconeogenesis & glycogen synthesis (transcriptional control)

Glucose and Electrons

• Cells are provided with a constant supply of NADPH for biosynthesis by the pentose phosphate pathway
• Ribose-5-P is also produced for RNA
• The pathway consists of two oxidative processes followed by five non-oxidative steps
• It works mostly in the cytosol of liver and adipose cells
• NADPH is used in the cytosol for fatty acid synthesis

Pentose Phosphate Pathway Stages

• It operates in liver and adipose tissue and generates NADPH and ribose-5-phosphate
• ATP is the cell's "energy" currency
• NADPH drives endergonic reductive biosynthesis
• It's an alternative pathway to glycolysis

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

Oxidative Reactions

• Reactions 1-3 facilitate NADPH production
• 3 Glucose-6-P + 6 NADP+ + 3 H2O yields 6 NADPH + 6 H+ + 3 CO2 + Ribulose-5-P

Isomerization and Epimerization

• Reactions 4 & 5 produce ribose-5-P for nucleic acid biosynthesis
• 3 Ribulose-5-P yields Ribose-5-P + 2 Xylulose-5-P

Bond Cleavage and Formation

• Reactions 6 – 8 convert Fructose-6-P & Glyceraldehyde-3-P to glycolysis
• Ribose-5-P + 2 Xylulose-5-P yields 2 Fructose-6-P + Glyceraldehyde-3-P

Oxidative Reactions

Glucose-6-Phosphate Dehydrogenase

• The products are NADPH and cyclic ester
• It's an irreversible first step that is highly regulated and inhibited by product NADPH

Reactions

• NADPH and cyclic ester is produced by the enzyme Glucose-6-Phosphate Dehydrogenase
• 6-Phosphogluconate Dehydrogenase enables oxidative decarboxylation and converts Product Ru5P to be Ru5P or Xu5P for further use

Isomerization and Epimerization Reactions

Phosphopentose Isomerase

• Enediol intermediate
• Product R5P used in nucleic acid biosynthesis and coenzymes (NADH, NADPH, FAD & B12

Phosphopentose Epimerase

• Enediol intermediate
• Inversion of –OH group at single carbon atom

Bond Cleavage and Formation Reactions

Transketolase

• Two carbon transfer from ketose to aldose

Transaldolase

• Three carbon transfer from ketose to aldose
• Converts sedoheptulose-7-P into products that can enter glycolysis.

Transketolase

• The whole pathway is reversible with the exception of the first step of Glycolysis
• Has an overall reaction of 3 C5 à C6 + C3

Pentose Phosphate Pathway Control

• Glucose-6-Phosphate is a crucial branch point in glycolysis and pentose phosphate pathway
• Flux through the pentose phosphate pathway and the rate of NADPH production are controlled at the first step through the glucose-6-phosphate dehydrogenase
• Energy is made by having glucose-6-P enter glycolysis
• Make NADPH or Ribose-5-P by having glucose-6-P enter pentose phosphate pathway

Possibilities for Pentose Phosphate Pathway:

• Both ribose-5-phosphate and NADPH are needed
• The first 4 reactions predominate to make R5P & NADPH
• Glu-6-P + 2 NADP+ + H2O --> Ribose – 5 – P + CO2 + 2 NADPH + 2 H+
• More ribose-5-phosphate than NADPH is needed, bypass oxidative reactions (1->4) to synthesize R5P without NADPH production and then 5 Glucose-6-P + ATP --> 6 Ribose-5-P + ADP + H+
• More NADPH than ribose-5-phosphate is needed and recycled by R5P, Glycolytic intermediates via gluconeogenesis so large amounts of
  • NADPH produced with the formula (Glu-6-P + 12 NADP+ 6 H2O -> 6 CO2 + 12 NADPH + 12 H+ + Pi)
• Both NADPH and ATP are needed, but ribose-5-phosphate is not then Glu-6-P --> Pyruvate