Metabolic Pathways: Glycolysis, TCA Cycle, and Electron Transport Chain

Overview

Review Topics: The lecture provides a review of phosphocreatine (PCr), glycolysis (including inhibition/stimulation), the glucose paradox, the tricarboxylic acid (TCA) cycle, and an introduction to $\beta$-oxidation.

GLUT-4 Translocation

Glucose Transport Stimulation: Glucose Transporter Type 4 (GLUT-4) is regulated by two main pathways:
- Insulin-Responsive Pathway:
  - Insulin binds to the insulin receptor, leading to tyrosine phosphorylation.
  - This activates IRS (Insulin Receptor Substrate) proteins.
  - IRS activates Phosphoinositide-3 Kinase (PI3K) via its SH2 domains (p110 and p85 subunits).
  - PI3K produces phosphatidylinositol (3,4,5)-bisphosphate (PIP3), which activates Phosphoinositide-dependent Kinases (PDKs).
  - PDKs activate Protein Kinase B (Akt).
  - Akt signals the translocation of insulin-responsive GLUT-4-containing vesicles to the cell membrane.
- Exercise-Stimulated Pathway:
  - Exercise activates 5'-AMP-activated Kinase (AMPK).
  - AMPK triggers the translocation of exercise-responsive GLUT-4-containing vesicles to the cell membrane.
Actual Translocation Process:
- Docking: GLUT-4 vesicles (containing v-SNARE proteins like synaptobrevin) dock with the cell membrane (containing t-SNARE proteins like syntaxin-4).
- Fusion: Once docked, the vesicles fuse with the cell membrane, inserting GLUT-4 transporters.
- Glucose Uptake: GLUT-4 facilitates the transport of glucose into the cell.
- Internalization and Re-sorting: After glucose uptake, GLUT-4 can be internalized via clathrin-coated vesicles, entering early endosomes, and then re-sorted back into vesicles, often involving insulin-responsive aminopeptidase.

Glycolysis

Process: Glycolysis is an anaerobic pathway that yields approximately $10\%$ of the available energy from a glucose molecule.
Key Steps and Regulatory Enzymes:
- Step 1: Glucose is converted to Glucose 6-phosphate. This is the first priming reaction, consuming ATP.
  - Enzyme: Hexokinase.
- Step 3: Fructose 6-phosphate converted to Fructose 1,6-bisphosphate. This is the second priming reaction, consuming ATP, and is the rate-limiting step.
  - Enzyme: Phosphofructokinase (PFK).
- Step 10: Phosphoenolpyruvate (2 molecules) converted to Pyruvate (2 molecules). This is the second ATP-forming reaction via substrate-level phosphorylation, producing $2\text{ ATP}$ per glucose.
  - Enzyme: Pyruvate Kinase (PK).
Regulation of Glycolysis:
- Hexokinase:
  - Inhibited by high ATP and Glucose 6-phosphate (G6P).
  - Stimulated by high glucose concentrations.
- Phosphofructokinase (PFK):
  - Inhibited by ATP, 1,6-bisphosphate (not 1,6, actually refers to Fructose 2,6-bisphosphate, an allosteric activator, or direct cellular ATP/AMP ratio), and citrate.
  - Stimulated by Fructose 6-phosphate (F6P), ADP, and AMP.
- Pyruvate Kinase (PK):
  - Activated by Fructose 1,6-bisphosphate (F1,6DP).

Glucose Paradox

Concept: The brain primarily utilizes glucose, but the liver paradoxically uses lactate (derived from glycolysis in other tissues like muscle and integument) to synthesize glycogen, rather than directly using glucose (especially after a meal, it can process glucose to glycogen but also convert dietary glucose to lactate and then use that lactate for glycogen synthesis, often in states of high glucose load when liver glycogen stores are full). Muscle and integument produce lactate from glucose, which can then be transported to the liver.
Circulation: Lactate and glucose levels are observed throughout the circulatory system (pulmonary artery, pulmonary vein, coronary sinus) to illustrate this metabolic fate.

Mitochondrion Structure and Function

Cellular Location: The mitochondrion is a key organelle for aerobic metabolism.
Structure:
- Outer membrane: Permeable to small molecules.
- Inner membrane: Highly folded into cristae; impermeable to most ions and small molecules; contains ATP synthase and components of the Electron Transport Chain (ETC).
- Intermembrane space: Space between the outer and inner membranes, where the proton gradient is established.
- Matrix: Innermost compartment, containing enzymes for the TCA cycle, $\beta$-oxidation, and pyruvate oxidation.
- Cristae: Infoldings of the inner membrane, increasing surface area for ETC and ATP synthesis.
- ATP Synthase: Enzyme complex located on the inner membrane, synthesizing ATP from ADP and $P_i$ .

Pyruvate to Acetyl-CoA Conversion

Process: Pyruvate, the end-product of glycolysis, is transported into the mitochondrial matrix.
Enzyme Complex: Pyruvate Dehydrogenase (PDH) Complex converts pyruvate (3C) into Acetyl-CoA (2C).
- Reaction: $\text{Pyruvate} + \text{NAD}^+ + \text{CoASH} \rightarrow \text{Acetyl-CoA} + \text{NADH} + \text{H}^+ + \text{CO}_2$
Significance: Acetyl-CoA is the hub of all aerobic metabolism, linking glycolysis to the TCA cycle and fatty acid oxidation.
Yield: From one glucose molecule, two pyruvates are produced, leading to two Acetyl-CoAs.
Regulation of PDH:
- PDH Kinase: Phosphorylates and inactivates PDH.
  - Stimulated by ATP/ADP ratio, NADH/NAD+ ratio, and Acetyl-CoA.
  - Inhibited by pyruvate and calcium ( $\text{Ca}^{2+}$ ) and insulin.
- PDH Phosphatase: Dephosphorylates and activates PDH.
  - Stimulated by calcium ( $\text{Ca}^{2+}$ ) and insulin.

Tricarboxylic Acid (TCA) Cycle (Krebs Cycle, Citric Acid Cycle)

Location: Primarily in the mitochondrial matrix, except for Succinate Dehydrogenase which is embedded in the inner mitochondrial membrane.
Purpose: To oxidize Acetyl-CoA to $CO_2$ and generate reducing equivalents (NADH and FADH2) for the ETC.
Key Steps and Enzymes:
- Entry: Acetyl-CoA (2C) combines with Oxaloacetate (OAA, 4C) to form Citrate (6C).
 - Enzyme: Citrate Synthase.
- Isocitrate Formation: Citrate is isomerized to Isocitrate (6C) via cis-Aconitate.
 - Enzyme: Aconitase.
- $\alpha$-Ketoglutarate Formation: Isocitrate is oxidized and decarboxylated to $\alpha$-Ketoglutarate (5C), producing $\text{NADH} + \text{H}^+$ and $CO2$ (first $CO2$ release).
 - Enzyme: Isocitrate Dehydrogenase.
- Succinyl-CoA Formation: $\alpha$-Ketoglutarate is oxidized and decarboxylated to Succinyl-CoA (4C), producing $\text{NADH} + \text{H}^+$ and $CO2$ (second $CO2$ release).
 - Enzyme: $\alpha$-Ketoglutarate Dehydrogenase Complex.
- Succinate Formation: Succinyl-CoA is converted to Succinate (4C), producing GTP (which can be converted to ATP).
 - Enzyme: Succinyl-CoA Synthetase.
 - GTP to ATP Conversion: $\text{GTP} + \text{ADP} \rightarrow \text{GDP} + \text{ATP}$ via nucleotide diphosphate kinase.
- Fumarate Formation: Succinate is oxidized to Fumarate (4C), producing $\text{FADH}_2$ .
 - Enzyme: Succinate Dehydrogenase (SDH) - unique to the TCA cycle as it's part of Complex II of the ETC.
- Malate Formation: Fumarate is hydrated to Malate (4C).
 - Enzyme: Fumarase.
- Oxaloacetate Regeneration: Malate is oxidized to Oxaloacetate (4C), regenerating OAA for the next cycle and producing $\text{NADH} + \text{H}^+$ .
 - Enzyme: Malate Dehydrogenase.
Role of Malate: Malate, once in the mitochondria, keeps the TCA cycle spinning by providing Oxaloacetate for Acetyl-CoA to bind to.
Important Cofactors: The cycle utilizes NAD+ and FAD, which are reduced to NADH + H+ and FADH2, respectively.
Regulation of the TCA Cycle:
- Inhibitors: Citrate, Acetyl-CoA, Succinyl-CoA, Pyruvate, ATP, NADH, Fatty acyl-CoA, acyl CoA derivatives of fatty acids, and high levels of pathway products.
- Activators: Ca $^{2+}$ , ADP, AMP, and substrate availability.

Monocarboxylate Transport (MCT)

Function: MCTs transport monocarboxylates like pyruvate and lactate across membranes (cytosol to mitochondrion and vice versa), often co-transporting $H^+$ . This is crucial for shuttle systems.

ATP Yield from Glucose Oxidation (Bookkeeping)

Equivalents (Common Values):
- $1\text{ NADH} + \text{H}^+ = 3\text{ ATP}$
- $1\text{ FADH}_2 = 2\text{ ATP}$
Overall ATP Yield:
- Glycolysis:
 - Direct Phosphorylation: $2\text{ ATP}$
 - $2\text{ NADH} + \text{H}^+ \times 3\text{ ATP/NADH} = 6\text{ ATP}$ . (Note: If Glycerol-3-phosphate shuttle is used, this may be $4\text{ ATP}$ from FADH2)
- Pyruvate Oxidation and TCA Cycle (per glucose molecule, so two Acetyl-CoAs):
 - $2 \times (4\text{ NADH} + \text{H}^+) \times 3\text{ ATP/NADH} = 24\text{ ATP}$ (This accounts for 2 NADH from PDH, and 6 NADH from TCA; $2 \times (1\text{ NADH from PDH} + 3\text{ NADH from TCA}) = 8\text{ NADH}$ , so $8\text{ NADH} \times 3\text{ ATP/NADH} = 24\text{ ATP}$ ).
 - $2 \times (1\text{ FADH}2) \times 2\text{ ATP/FADH}2 = 4\text{ ATP}$
 - $2 \times (1\text{ direct phosphorylation (GTP)}) = 2\text{ ATP}$
Total ATP from Glucose Oxidation: Summing up, the total yield is approximately $38\text{ ATP}$ .
The 36-38 ATP Difference:
- The variation arises from the shuttle system used to transport cytosolic NADH into the mitochondria for the ETC.
- Glycerol Phosphate Shuttle (e.g., in insects): Costs $2\text{ ATP}$ equivalents because it transfers electrons to FADH2 (which yields $2\text{ ATP/FADH}_2$ instead of $3\text{ ATP/NADH}^+$ ), leading to a net of $36\text{ ATP}$ .
- Malate-Aspartate Shuttle (e.g., in humans): Costs $0\text{ ATP}$ because it effectively regenerates NADH in the mitochondria, leading to a net of $38\text{ ATP}$ .

Shuttle Systems (Transport of Reducing Equivalents)

Purpose: To transport NADH (reducing equivalents) generated in the cytosol (e.g., from glycolysis) into the mitochondrial matrix, as NADH cannot directly cross the inner mitochondrial membrane.
1. Malate-Aspartate Shuttle:
- Cytosol: Cytosolic NADH reduces Oxaloacetate (OAA) to Malate via cytosolic Malate Dehydrogenase (MDH1).
- Malate is transported into the mitochondrial matrix (via SLC25A11, the malate-alpha-ketoglutarate carrier).
- Mitochondria: Mitochondrial Malate Dehydrogenase (MDH2) oxidizes Malate back to OAA, regenerating NADH within the matrix for the ETC.
- To complete the cycle, OAA is transaminated to Aspartate (via mitochondrial GOT2), which exits the mitochondrion (via SLC25A13), is converted back to OAA in the cytosol (via cytosolic GOT1), completing the cycle.
- This shuttle preserves the full energy yield of NADH ( $3\text{ ATP}$ /NADH).
2. Glycerol-3-Phosphate Shuttle:
- Cytosol: Cytosolic NADH reduces Dihydroxyacetone Phosphate (DHAP) to Glycerol-3-phosphate via cytosolic Glycerol-3-Phosphate Dehydrogenase (cGPSDH).
- Glycerol-3-phosphate then transfers electrons to FAD (part of mitochondrial Glycerol-3-Phosphate Dehydrogenase, mGPDH, located on the outer surface of the inner mitochondrial membrane), reducing FAD to FADH2.
- FADH2 then passes its electrons directly to the ETC (Coenzyme Q).
- This shuttle effectively transfers electrons from cytosolic NADH to mitochondrial FADH2, resulting in $2\text{ ATP}$ /NADH equivalent instead of $3\text{ ATP}$ .

Key Points of the TCA Cycle

Energy Conservation: Five sites of energy conservation through the production of reduced cofactors (NADH and FADH2).
Direct Phosphorylation: One "direct" phosphorylation step (GTP converted to ATP).
ATP Yield: Yields $38\text{ ATP}$ per glucose (sometimes $36\text{ ATP}$ depending on the shuttle).
Regulation: Inhibited by compounds that are products of the pathway (e.g., ATP, NADH, Succinyl-CoA).

Electron Transport Chain (ETC)

Mechanism:
- Receives electron pairs from reduced cofactors (NADH and FADH2) generated by glycolysis, pyruvate oxidation, and the TCA cycle.
- Electrons are passed along a series of protein complexes (Complex I, II, III, IV) embedded in the inner mitochondrial membrane.
- This electron flow powers the pumping of $H^+$ ions from the mitochondrial matrix to the intermembrane space, generating an electrochemical proton gradient ( $\Delta\Psi$ ).
- Oxygen ( $O2$ ) is the final electron acceptor, forming water ( $H2O$ ).
ATP Synthesis: The $H^+$ ions flow back into the matrix through ATP synthase, driving the synthesis of ATP from ADP and $P_i$ (oxidative phosphorylation).
Energy Conservation: The ETC conserves the chemical energy in foodstuffs into chemical energy useful to the cells (ATP).

Measuring Respiratory Rate

Methodology: Oxygen consumption (rate of electron flow) is measured in a closed system containing mitochondria (mito).
Phases:
- Initial Baseline (without electron donors or ADP): Oxygen is at $100\%$ (saturated oxygen), little to no respiration.
- Substrate Addition (P+M, e.g., pyruvate and malate): Adding electron donors (substrates like pyruvate and malate) initiates some basal respiration (oxygen consumption decreases).
- ADP Addition: Adding ADP significantly increases the rate of oxygen consumption (respiration). This is State III respiration, where both substrate and ADP are abundant, allowing rapid ATP synthesis.
- ADP Depletion: Once ADP is consumed and converted to ATP, the rate of respiration slows down again (oxygen consumption decreases but not to zero). This is State IV respiration, limited by the availability of ADP.
Significance: The rate of respiration (electron flow) is determined by the rate at which protons enter back into the matrix, which is linked to ADP availability.

Training and Mitochondria

Training Adaptations:
- Causes large changes in mitochondrial enzymes, increasing mitochondrial volume and mass (mitochondrial biogenesis).
- More mitochondria lead to a greater capacity to respond to changes in ADP.
- Effect on ADP: Trained individuals exhibit lower ADP levels compared to untrained individuals during exercise.
- Impact on Glycolysis and Lactate: Lower ADP attenuates (reduces) the rate of glycolysis and lactate formation, as PFK is stimulated less.
- Glycogen Sparing: This leads to less glycogen utilization, sparing glycogen stores, which is a classic training effect.
- Endurance: Spared glycogen allows individuals to run longer in endurance activities like marathons.
- Reactive Oxidative Species (ROS): More electron transport system (ETS) components due to increased mitochondria also lead to less production of Reactive Oxidative Species (ROS), as electrons are passed more efficiently, reducing