Week 13 - Glucose-6-phosphate: a shared metabolite; Glycolysis vs glyconeogenesis

Lecture 1: Glucose-6-phosphate: a shared metabolite

1. Why G6P matters

Glucose‑6‑phosphate (G6P) is the first committed intracellular form of glucose and acts as a metabolic junction.
Once glucose → G6P (via hexokinase), it can no longer leave the cell (charged phosphate).
G6P can be directed into:

1. Glycolysis → ATP + pyruvate

2. Pentose phosphate pathway (PPP) → NADPH + ribose‑5‑P

3. Glycogen synthesis → glucose storage

4. G6P → glucose (liver only) via G6Pase

5. Glycerol backbone synthesis (via DHAP)

6. Indirectly → fatty acid synthesis (via acetyl‑CoA)

Flux depends on ATP/ADP/AMP, NADH/NAD⁺, NADPH/NADP⁺, and hormonal signals.

2. Global metabolic logic

Energy charge controls everything

• High ATP / high NADH → slow glycolysis + TCA

• High ADP/AMP / high NAD⁺ → accelerate glycolysis + TCA

• Citrate (from TCA) inhibits PFK‑1 → coordinates glycolysis with TCA capacity

• G6P inhibits hexokinase → prevents unnecessary glucose phosphorylation

• Acetyl‑CoA activates pyruvate carboxylase → directs carbon toward gluconeogenesis/fat synthesis

3. Dietary carbohydrate entry points

Different sugars feed into glycolysis at different points:

All converge on G6P or F6P.

4. Why glycolysis has >1 irreversible steps

Irreversible steps = control points.
There are three because glycolysis has multiple branch points where carbon can leave or enter:

1. Hexokinase (Glucose → G6P)

   • Controls glucose entry

   • G6P can go to PPP, glycogen, glycolysis

2. PFK‑1 (F6P → F1,6BP)

   • Major flux‑determining step

   • DHAP can be diverted to glycerol/fat synthesis

3. Pyruvate kinase (PEP → pyruvate)

   • Controls carbon entering pyruvate fates (lactate, acetyl‑CoA, gluconeogenesis)

Each irreversible step sits before or after a major metabolic branch, allowing independent regulation of each segment.

5. Pentose Phosphate Pathway (PPP)

Purpose

PPP has two major outputs:

(1) NADPH

Used for:

• Fatty acid synthesis

• Cholesterol/steroid synthesis

• Glutathione recycling (antioxidant defence)

(2) Ribose‑5‑phosphate

Used for:

• Nucleotide synthesis (DNA/RNA)

• Coenzymes (NAD⁺, FAD, CoA)

Oxidative phase

G6P → 6‑phosphogluconate → ribulose‑5‑P
Produces 2 NADPH + CO₂

Non‑oxidative phase

Interconverts C3/C4/C5/C6/C7 sugars
Allows:

• Ribose production without NADPH

• NADPH production without ribose

• Recycling back to glycolysis (F6P, GAP)

Control

• High NADPH inhibits G6PD (first enzyme)

• Ensures PPP only runs when NADPH is needed

6. Glycogen — structure & purpose

What is glycogen?

• Highly branched polymer of glucose

• α(1→4) chains with α(1→6) branches

• Stored mainly in liver and skeletal muscle

Why branched?

• Many non‑reducing ends → rapid release of glucose

• Essential for fast ATP demand (muscle)

• Liver uses glycogen to maintain blood glucose during fasting

7. Glycogen synthesis (anabolic pathway)

Stepwise

1. G6P → G1P
   Phosphoglucomutase

2. G1P + UTP → UDP‑glucose
   UDP‑glucose pyrophosphorylase
   (Activation step; PPi hydrolysis drives reaction)

3. UDP‑glucose → glycogen (elongation)
   Glycogen synthase

   • Adds glucose to non‑reducing ends

   • Forms α(1→4) linkages

4. Branching enzyme

   • Transfers 6–7 residue segments

   • Creates α(1→6) branch points

Regulation

• Glycogen synthase active when dephosphorylated

• Activated by insulin, inhibited by glucagon/epinephrine

8. Glycogen breakdown (catabolic pathway)

Stepwise

1. Glycogen → G1P
   Glycogen phosphorylase

   • Cleaves α(1→4) bonds

   • Uses Pi (not ATP)

2. Debranching enzyme

   • Transferase moves 3 residues

   • α(1→6) glucosidase releases free glucose

3. G1P → G6P
   Phosphoglucomutase

Tissue differences

• Muscle: G6P enters glycolysis (no G6Pase)

• Liver: G6P → glucose via G6Pase → blood glucose

Regulation

• Phosphorylase active when phosphorylated

• Activated by:

  • Glucagon (liver)

  • Epinephrine (muscle)

  • Ca²⁺ (muscle contraction)

  • AMP (low‑energy muscle)

9. Hormonal control: insulin vs glucagon

Insulin (fed state)

• ↓ phosphorylation of metabolic enzymes

• Activates:

  • Glycogen synthase

  • PFK‑1 (via ↑F2,6BP)

  • Hexokinase II

• Inhibits:

  • Glycogen phosphorylase

  • Phosphorylase kinase

→ Promotes glycogen synthesis + glycolysis

Glucagon (fasted state)

• ↑ phosphorylation

• Activates:

  • Glycogen phosphorylase

  • Phosphorylase kinase

• Inhibits:

  • Glycogen synthase

  • PFK‑2 (↓F2,6BP → ↓PFK‑1)

→ Promotes glycogen breakdown + gluconeogenesis

10. Liver‑specific: G6P → glucose

Only the liver (and kidney cortex) express glucose‑6‑phosphatase.

Process:

1. G6P transported into ER lumen

2. G6Pase removes phosphate → glucose

3. Glucose exported to blood via GLUT2

Purpose:

• Maintain blood glucose during fasting

• Support brain + RBCs (obligate glucose users)

11. Integrated logic

Fed state (high glucose)

• High ATP → slow glycolysis

• High citrate → slow PFK‑1

• G6P accumulates → glycogen synthesis + PPP

• Excess acetyl‑CoA → fatty acid synthesis

Fasted state

• Low ATP → activate glycolysis

• Low blood glucose → glucagon → glycogen breakdown

• Liver releases glucose to bloodstream

12. Key enzymes to remember

• G6PD (PPP control)

• Glycogen synthase (storage)

• Glycogen phosphorylase (release)

• Phosphoglucomutase (G1P ↔ G6P)

• UDP‑glucose pyrophosphorylase (activation step)

Lecture 2: Glycolysis vs. gluconeogenesis

1. Why gluconeogenesis exists

Problem: Humans do not eat continuously, but many tissues require continuous glucose (especially CNS + RBCs).
Solution: The liver (and kidney during prolonged fasting) synthesises glucose from non‑carbohydrate precursors.

Key points

• Glycolysis is universal (all cells).

• Gluconeogenesis is restricted to liver and kidney.

• Liver must:

  • Use glucose when fed

  • Produce glucose when fasted

  • Avoid futile cycling (glucose → pyruvate → glucose)

2. Glycolysis vs Gluconeogenesis: Antagonistic Pathways

These pathways run in opposite directions and share many reversible steps.

Irreversible steps of glycolysis (must be bypassed):

1. Hexokinase (Glucose → G6P)

2. PFK‑1 (F6P → F1,6BP)

3. Pyruvate kinase (PEP → Pyruvate)

Gluconeogenic bypass enzymes

Energetics:

• Glycolysis net: +2 ATP

• Gluconeogenesis cost: 6 ATP equivalents
  → Strong need for reciprocal regulation to prevent futile cycling.

3. Pyruvate → PEP: the hardest reversal

Because pyruvate kinase is strongly irreversible, gluconeogenesis uses two steps:

Pathway A (common): mitochondrial route

1. Pyruvate → OAA (pyruvate carboxylase, uses ATP)

2. OAA → malate (to export reducing equivalents)

3. Malate → OAA (cytosol)

4. OAA → PEP (PEPCK, uses GTP)

Pathway B (when lactate high): cytosolic route

• Lactate → pyruvate → OAA → PEP

• Avoids need to shuttle NADH out of mitochondria.

4. Liver‑specific adaptations enabling gluconeogenesis

4.1 Glucose transport: GLUT2

• High Km (~17 mM) → low affinity

• Only imports glucose when blood glucose is high

• Allows glucose to exit during fasting without being re‑captured

• Makes liver a glucose buffer.

4.2 Hexokinase isoform: Glucokinase (HK4)

• High Km (~10 mM) → only active when glucose is abundant

• Prevents liver from trapping glucose as G6P during fasting

• Regulated by nuclear sequestration:

  • High glucose → HK4 released into cytosol → glycolysis ON

  • High F6P → HK4 pulled into nucleus → glycolysis OFF

5. The master control switch: Fructose‑2,6‑bisphosphate (F26BP)

F26BP is not a glycolytic intermediate.
It is a regulatory metabolite that coordinates glycolysis vs gluconeogenesis.

Effects

• ↑ F26BP → activates PFK‑1 → glycolysis ON

• ↓ F26BP → activates FBPase‑1 → gluconeogenesis ON

Made & destroyed by the same bifunctional enzyme

PFK‑2 / FBPase‑2

• Phosphorylation state determines which activity dominates.

Hormonal control

6. Pyruvate kinase regulation (liver‑specific)

• Inhibited by ATP, acetyl‑CoA, long‑chain fatty acids (signals of high energy)

• Glucagon → PKA → phosphorylates pyruvate kinase → inactivates it
  → Prevents futile cycling during gluconeogenesis.

7. Substrate availability for gluconeogenesis

Major carbon sources:

• Lactate (Cori cycle)

• Alanine (muscle proteolysis)

• Glycerol (lipolysis)

• TCA intermediates (via OAA)

Acetyl‑CoA cannot be converted to glucose, but:

• High acetyl‑CoA activates pyruvate carboxylase

• Signals liver to run gluconeogenesis using fat oxidation as fuel.

8. Fed vs Fasted metabolic states

Fed state (high insulin)

• Glycolysis ↑

• Glycogen synthesis ↑

• Fatty acid synthesis ↑

• Gluconeogenesis ↓

• F26BP ↑

• Pyruvate kinase active

• HK4 active in cytosol

Fasted state (high glucagon)

• Glycolysis ↓

• Glycogen breakdown ↑

• Gluconeogenesis ↑

• F26BP ↓

• Pyruvate kinase phosphorylated (inactive)

• HK4 sequestered in nucleus

• Liver exports glucose

9. Why futile cycling must be avoided

If glycolysis and gluconeogenesis ran simultaneously:

Glucose → pyruvate → glucose
= Net loss of 4 ATP per cycle
→ Catastrophic energy drain.

Thus:

• Hormonal control

• Allosteric control

• Enzyme phosphorylation

• Substrate availability
  all ensure only one pathway dominates at a time.

10. Integration with whole‑body physiology

• Liver acts as glucose reservoir for CNS + RBCs.

• Uses fatty acid oxidation to power gluconeogenesis.

• Muscle does not perform gluconeogenesis (lacks G6Pase).

• Kidney contributes during prolonged fasting.

11. Key exam‑level takeaways

• Know the three irreversible glycolytic steps and their gluconeogenic bypasses.

• Understand F26BP as the central reciprocal regulator.

• Liver uses GLUT2 and HK4 to allow glucose export.

• Glucagon → ↓F26BP → gluconeogenesis ON.

• Energetics: gluconeogenesis is expensive, requiring 6 ATP equivalents.

• Pyruvate → PEP requires two enzymes and mitochondrial–cytosolic coordination.

• Acetyl‑CoA activates pyruvate carboxylase but cannot become glucose.