Gluconeogenesis: Glucose Synthesis

• Gluconeogenesis is "making new glucose".

Dependence on Glucose

• Cells rely on glucose for energy.
• Brain and red blood cells are almost entirely dependent on glucose.
• Liver-stored glycogen can supply the brain with glucose for only 1½ days when fasting.
• If the body cannot obtain glucose from diet, it produces it from non-carbohydrate precursors.

What Gluconeogenesis Is

• Gluconeogenesis is "the generation of new sugar."
• New glucose is synthesized from lactate, pyruvate, most amino acids through protein hydrolysis, and glycerol through TAG hydrolysis.
• Animals cannot use acetyl CoA to synthesize glucose.
• Plants use the glyoxalate cycle with acetyl CoA to make glucose.
• Gluconeogenesis occurs in the liver (90%) and kidneys (10%).
• Microorganisms use acetate and propionate as gluconeogenesis substrates.

Cori Cycle

• Lactate and pyruvate are essential within the Cori Cycle

How Glycolysis & Gluconeogenesis Relate

• Gluconeogenesis and glycolysis are opposing metabolic pathways with three differences:
• 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 → 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 → Glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
• Adding both net reactions together: 2 ATP + 2 GTP + 4 H2O → 2 ADP + 2 GDP + 4 Pi
• Gluconeogenesis tightly regulated, wasteful cycle.
• Gluconeogenesis enzymes are in the cytosol, except glucose-6-phosphatase, which is in the ER, and pyruvate carboxylase, which is in the mitochondria.

Unique Reactions of Gluconeogenesis

• Bypass I: Pyruvate → Oxaloacetate → Phosphoenolpyruvate; replacing pyruvate kinase.
• Pyruvate Carboxylase – Carboxylation happens in the mitochondrial matrix.
• The reaction is: Pyruvate + HCO3- + ATP → Oxaloacetate + ADP + Pi
• Acetyl CoA activates
• Cofactor is Biotin
• Oxaloacetate is too polar to cross mitochondrial membranes, so it is converted to aspartate or malate for transport.
• Phosphoenolpyruvate Carboxykinase - Decarboxylation
• Occurs in the cytosol.
• Oxaloacetate, a 'high energy' intermediate, allows for the exergonic decarboxylation needed to form PEP.
• Beta-ketoacids are 'high energy' intermediates because the loss of CO2 generates a strong enolate.
• Pyruvate carboxylase is a biotin-dependent enzyme.
• Bicarbonate must be activated for attack by the pyruvate carbanion, driven by ATP.
• Bypass II: Fructose 1,6-Bisphosphate → Fructose 6-Phosphate
• Replaces phosphofructokinase
• Fructose 1,6-Bisphosphatase - Phosphoester hydrolysis
• Occurs in cytosol.
• Bypass III: Glucose 6-Phosphate → Glucose
• Replaces hexokinase
• Glucose 6-Phosphatase - Phosphoester hydrolysis
• Occurs in ER
• The enzyme is not found in muscle or brain, meaning gluconeogenesis cannot occur in these organs.
• Glucose is exported from the ER into the bloodstream, going to the brain to carry out glycolysis.

How Gluconeogenesis is Regulated

• Reciprocal Control of Glycolysis & Gluconeogenesis.
• Glycolysis is ON when the cell needs ATP & NADH.
• Gluconeogenesis is ON when the cell does not need ATP & NADH and there is excess lactate/pyruvate, and the cell wants to store glucose as glycogen.
• Mechanisms of regulations:
• Substrate-level control of Gluocose-6-phosphatase occurs.
• Allosteric regulation via Fructose-2,6,-Bisphosphate & Acetyl CoA occurs.
• Covalent modification, phosphorylation/dephosphorylation, via cAMP allows for hormonal control.
• Transcriptional control of the gene encoding PEP carboxykinase occurs.
• Phosphorylation inhibits gluconeogenesis and stimulates glycolysis.

Glycogen

• Body obtains glucose from storage or dies
• Glycogen is mostly stored in the liver and muscles.
• The structure is optimized to store and deliver energy quickly and for the longest time.
• Key for optimization is average branch chain length of 13 residues

Glycogen & Starch Catabolism

• Alpha-Amylase is an endoglycosidase α(1->4).
• It is active on either side of a branch point, but its activity is reduced near branch points.
• Debranching enzyme cleaves "limit dextrins" and has two activities:
• Transferring trisaccharide groups.
• Cleaving remaining single glucose units from the main chain α(1->6).

Tissue Glycogen Metabolism Regulation

• Digestive breakdown of starch is unregulated - nearly 100% of ingested food is absorbed and metabolized.
• Tissue glycogen is a breakdown that is carefully controlled with high MW "granules" and it its important for energy
• Glycogen phosphorylase cleaves glucose from the nonreducing ends of glycogen molecules with phosphate as nucleophile in a "phosphorolysis", not a hydrolysis
• Product is a sugar-P - a potential glycolysis substrate.

Glycogen Catabolism

• Glycogen Phosphorylase
• It liberates glucose units from stored glycogen reserves in muscles & liver when energy is needed
• Tightly regulated via allosteric regulation & covalent modification, it is paradigm of enzyme regulation
• It is activated when cell needs energy and inactivated when cells at rest (store glucose as glycogen)
• Catalytic Reaction
• This Cleaves glucose units from non reducing ends of glycogen Phophorolysis not hydrolysis for glucose-1-phosphate product

Glycogen Phosphorylase Regulation

• Allosteric Effectors follow a meets normal metabolic needs , under normal conditions as positive homotropic effector: Pi Negative heterotropic effectors: ATP & glucose-6-P and positive heterotropic effector: AMP
• Covalent Modification emergency situation
• occurs in "fight or flight" situations and covalent modification overrides everything
• more active phosphorylase a phosphorylation of Ser-14
• hormone-activated signal cascade (glucagon & epinephrine)

Glycogen Synthesis

• Higher animals store excess glucose as glycogen
• Glycogen phosphorylase (Glycogen → Glucose) Vs glycogen synthase (Glu → Gly)
• Activation of glucose by formation of nucleotide sugars
• Sugar Nucleotides activated forms of sugar units
• UDP-glucose – donor for glycogen biosynthesis
• Nucleotide labels mark sugar
• ADP-glucose – donor for starch biosynthesis
• UDP-glucose Pyrophosphorylase
• Reaction is driven by PPi hydrolysis
• Only one phosphoanhydride bond is consumed in the storage of one glucose in glycogen. Upon glycogen degradation, one glucose yields 32 ATP

Glycogen Synthesis (Continued)

• Synthesis of glycogen via glycogen synthase and branch enzyme: UDP-Glucose + (Glucose)n → UDP + (Glu)n+1
• Glucose is added to the non-reducing ends of glycogen by glycogen synthase.
• Glycogenin, a protein, is covalently linked to the first glucose via an acetal linkage to Tyr-OH.
• α (1→6) branches every 8-12 residues generate more non-reducing ends.

Glycogen Metabolism Control

• Hormones regulate glycogen metabolism & blood glucose levels
• Insulin (lowers blood glucose) and Glucagon (raises) maintain blood glucose levels around 4.5 mM.
• Glycogen breakdown is triggered by glucagon & epinephrine
• Glycogen biosynthesis by insulin
• Cortisol & Glucocorticoid cause long-term stress hormones that affects liver, skeletal muscle and adipose tissues
• Promotes protein breakdown & decreases protein synthesis.
• Stimulates gluconeogenesis & Glycogen synthesis (transcriptional control).

Glucose Provide Electrons for Biosynthesis

• Cells are provided with a constant supply of NADPH for biosynthesis by the pentose phosphate pathway
• This pathway produces ribose-5-Pathway for RNA
• It operates mostly in cytosol of liver and adipose cells
• It uses NADPH for fatty acid

Electron Biosynthesis

• Pentose phosphate pathway operates in liver & adipose tissue.
• Generates NADPH (reducing power) & ribose-5-phosphate
• ATP is the cell's "energy" currency. NADPH is used to drive endergonic reductive biosynthesis.
• Pentose phosphate pathway is an alternative pathway to glycolysis.
• Overall Net Reaction: 3 Glucose-6-P + 6 NADP+ + 3 H2O ⇔ 6 NADPH + 6 H+ + 3 CO2 + 2 Fructose-6-P + Glyceraldehyde-3-P
• Three Stages of Pentose Phosphate Pathway:

1. Oxidative Rxns: Rxns 1 – 3: NADPH production: 3 Glucose-6-P + 6 NADP+ + 3 H2O → 6 NADPH + 6 H+ + 3 CO2 + Ribulose-5-P
2. Isomerization & Epimerization Rxns: Rxns 4 & 5: Ribose-5-P is for nucleic acid biosynthesis: 3 Ribulose-5-P ⇔ Ribose-5-P + 2 Xylulose-5-P
3. C–C Bond Cleavage & Formation Rxns: Rxns 6 – 8: Fructose-6-P & Glyceraldehyde-3-P is for glycolysis: Ribose-5-P + 2 Xylulose-5-P ⇔ 2 Fructose-6-P + Glyceraldehyde-3-P

Oxidative Reactions

• Glucose-6-Phosphate Dehydrogenase yields NADPH & cyclic ester
• Irreversible 1st step & highly regulated. Inhibited by product NADPH
• Gluconolactonase (hydrolysis) and 6-Phosphogluconate Dehydrogenase.
• Product Ru5P must be converted to R5P or Xu5P for further use

Isomerization & Epimerization Rxns

• Phosphopentose Isomerase, Enediol intermediate
• Product R5P used in nucleic acid biosynthesis (NADH, NADPH, FAD & B12)
• Phosphopentose Epimerase (Proton Transfer) with Enediol intermediate and inversion of -OH group

Reactions for C–C Bond Cleavage & Formation

• Ketolase and Transaldolase are involved
• Transketolase
• Two carbon transfer from ketose to aldose
• Transaldolase that converst sedohepatulose-7-P using products for glycolysis
• Three carbon transfer from ketose to aldose
• Transketolase

Pentose Phosphate Pathway

• Glucose-6-Phosphate is a crucial branch point in glycolysis & pentose phosphate pathway
• Its production controls production controlled at 1st step through glucose-6-phosphate dehydrogenase
• Make ATP by having glucose-6-P enter glycolysis or NADPH for by having glucose-6-P enter pentose phosphate pathway.
• Four Possibilities for Pentose Phosphate Pathway:
• Both ribose-5-phosphate & NADPH if needed
• More ribose-5-phosphate than NADPH if needed through bypassing oxidation reactions
• More NADPH than ribose-5-phosphate if needed through recycling R5P to become glycolytic
• Both NADPH & ATP are needed if ribose-5-phosphate is not needed and converts to pyruvate