Glycolysis & Pyruvate Dehydrogenase

Glucose

- The form of sugar that circulates in the blood and provides the major source of energy for body tissues. When its level is low, we feel hunger.

Carbohydrates

- The starches and sugars present in foods, broken down into glucose to provide energy

Common Formula for Carbohydrates

- Cn(H2O)n

- "Watered carbon"

Glucose: Formula

C6H12O6

Monosaccharides

- The simplest carbohydrates

Key Monosaccharides

- Glucose, Fructose, Galactose

Glucose, Fructose, Galactose: How are they similar? How are the different?

- Similar: All have the same chemical formula C6H12O6

- Different: They are isomers with different spacial orientations

Disaccharides

- Carbohydrates that are made up of two monosaccharides

What happens to disaccharides in the GI tract?

- Broken down into monosaccharides → then absorbed

Lactose

- Galactose + glucose

What breaks down Lactose? What happens when someone is lacking in this enzyme?

- Lactase

- Lactose intolerance

Sucrose

- Glucose + fructose, found in table sugar

What breaks down Sucrose?

- Sucrase

Polysaccarides

- Polymers of monosaccharides

Common Polysaccharides

- Starch

- Glycogen

- Cellulose

Starch

- A storage polysaccharide in plants consisting entirely of glucose.

Glycogen

- An extensively branched glucose storage polysaccharide found in the liver and muscle of animals; the animal equivalent of starch.

Cellulose

- A substance (made of sugars) that is common in the cell walls of many organisms, cannot be broken down by animals

- "Fiber" in diet → improved bowel function

How can glucose be used in the body?

ATP production, amino acid synthesis, glycogen synthesis, triglyceride synthesis

Which organ has the most varied use of glucose?

Liver

- TCA for ATP

- Glycogen synthesis

How does the brain use glucose?

- Constant use of glucose for TCA cycle (ATP)

- Little glycogen storage

How do muscles/heart use glucose?

- Transport of glucose into cells is heavily influenced by insulin; more insulin → more glucose uptake

- TCA (ATP) and can store glucose as glycogen

How do RBCs use glucose?

- Only use glucose for anaerobic metabolism (generates ATP) because they lack mitochondria → generates lactate

How does Adipose Tissue use glucose?

- Converts glucose into fatty acids

- Uptake is influenced by insulin

How does glucose enter cells?

- Na+ independent entry

- Na+ dependent entry

Na+ Independent Entry

- GLUT-1 to GLUT-14 (uses [c] gradient)

- Varies by tissue (RBCs use GLUT-1)

Na+ Dependent Entry

- Glucose is absorbed from low [c] → high [c]

- Intestinal epithelium and renal tubules

How is Glucose Absorbed By Intestinal Epithelial Cells?

1. In GI lumen (left), you have low [glucose] compared to inside the cell

2. In order to absorb glucose, the cell will couple that absorption with sodium to move against [c] gradient (SGLT1, Na+ dependent)

3. [glucose] inside cell is higher than in interstitium/blood, so glucose can move down [c] gradient via GLUT2 (Na+ independent) into interstitium

4. Sodium previously uptaken by SGLT into cell (coupled to glucose) is pumped back out into GI Lumen to create high [Na+] outside of cell; this sodium can then be reused as a cotransporter for more glucose

GLUT-1: Insulin dependent or independent? Its result? Where is it utilized?

- Insulin Independent → uptake of glucose when [c] is high, less uptake when [c] is low (no influence from insulin)

- Location: Brain and RBCs

GLUT-4: Insulin dependent or independent? Its result? Where is it utilized?

- Insulin Dependent

- Fat tissue, skeletal muscle

GLUT-2: Insulin dependent or independent? Its result? Where is it utilized?

- Insulin independent

- Liver, Kidneys, Intestines, Pancreas

What is the directionality of GLUT-2?

- Bidirectional: Can transport glucose OUT of cells for gluconeogenesis

Which organs synthesize glucose for the body via gluconeogenesis?

- Liver and Kidney → they have GLUT-2 (bidirectional) so they can take up glucose but also put it back into bloodstream

What role does GLUT-2 have in Intestinal Cells?

- Transports glucose OUT of epithelial cells (from lumen) → portal vein

Glycolysis

- The breakdown of glucose by enzymes, releasing energy and pyruvic acid.

Which cells use glycolysis? Where does it occur within cells?

- ALL cells of the body

- Sequence of rxns occur in cytoplasm

What conversion happens in glycolysis? What does it generate?

- Converts: Glucose (6 carbons) → private (3 carbons)

- Generates: ATP and NADH

NADH

- The reduced form of NAD+; an electron-carrying molecule that functions in cellular respiration

- Generated during glycolysis

Cellular Respiration

- Process that releases energy by breaking down glucose and other food molecules in the presence of oxygen

NAD+ vs NADH

- NAD+: Accepts electrons

- NADH: (Made during glycolysis) Dontaes electrons to ETC → generates ATP

Are the reactions of glycolysis mostly reversible or irreversible? What does this allow?

- Majority of rxns are reversible

Allows for:

- When the level of one substrate gets high, ex. 3-phosphoglycerate, more 2-phosphoglycerate is generated

- On the other hand if [2-phosphoglycerate] is high, more 3-phosphoglycerate would be generated

MEANS RXNS CAN BE USED IN BOTH GLYCOLYSIS AND GLUCONEOGENESIS

Which Reactions of Glycolysis are Irreversible?

1. Glucose → Glucose-6-Phosphate

2. Fructose-6-Phosphate → Fructose-1,6-Bisphoshate

3. Phosphoenolpyruvate (PEP) → Pyruvate

What are the stages of Glycolysis?

1. Priming/Investment stage: Where the cell invests energy (spends ATP) to convert glucose to a more favorable structure to go through remaining rxns

2. Splitting Stage: The 6-carbon structure is divided into 2x 3-carbon structures

3. Energy Stage: Where ATP is generated

Glucose → Glucose-6-Phosphate: What enzyme catalyzes this rxn?

- Hexokinase

- Glucokinase

Hexokinase: Which rxn does it catalyze? Where is it found? Insulin dependent/independent?

- Rxn: Glucose → Glucose-6-Phosphate (Uses ATP)

- Location: Most Tissues

- Insulin Independent

What is Hexokinase Inhibited by? Its result?

- Inhibited by G-6-P → Prevents cells from hoarding glucose

High [G-6-P] means cells have satisfied their metabolic requirements, meaning it doesn't need more glucose → prevents further rxns from Hexokinase

Hexokinase: Kinetics (Km/Vm)

- Low Km: High affinity for glucose. It binds glucose very readily, even when glucose concentration is low.

- Low Vm: Even if you give it tons of glucose, it has a relatively low maximum rate at which it can process it.

Glucokinase: Which rxn does it catalyze? Where is it found? Insulin dependent/independent?

- Rxn: Glucose → Glucose-6-Phosphate (Uses ATP)

- Location: Liver/Pancreas

- Induced by Insulin

What is Glucokinase Inhibited by? Inhibition is overcome by what?

- Fructose-6-Phosphate

- Inhibition overcome by ↑↑ [Glucose]

When is Glucokinase inactive?

- In a fasting state → ↓ [Glucose] and ↑ [F6P]

- F6P is high because pyruvate is undergoing gluconeogenesis → you want to shut down glucokinase to prevent further processing of glucose

Glucokinase: Kinetics (Km/Vm)

High Km:

- Glucokinase doesn't bind glucose very well when glucose levels are low.

- It becomes much more active when there's lots of glucose around, like after a meal

High Vm:

- Once glucose is abundant, glucokinase can process large amounts of glucose quickly.

How does F6P Inactivate Glucokinase? How does Glucose overcome this inactivation?

1. F-6-P encourages Glucokinase Regulatory Protein to bind to glucokinase → translocated to the nucleus → inactivation of enzyme → no more glycolysis

2. When [Glucose] ↑↑ → Competes with GKRP for GK binding → GK goes back to cytosol where it can participate in glycolysis to generate ATP/NADH

Hexokinase vs Glucokinase: What is their activity in the setting of Low blood sugar? Its result?

- Hexokinase: Active (No inhibition by G6P)

- Glucokinase: Inactive (not working when ↓ [Glucose])

GLUCOSE GOES TO TISSUES FOR USE, NOT LIVER

Hexokinase vs Glucokinase: What is their activity in the setting of High blood sugar? Its result?

- Hexokinase: Inactive (inhibited by G6P)

- Glucokinase: Active (↑ [glucose] → glucokinase freed from nucleus)

GLUCOSE GOES TO LIVER TO BE STORED AS GLYCOGEN

Glucokinase Deficiency

- If deficient → less uptake of glucose by liver/pancreas → mild hyperglycemia

Glucokinase Deficiency is exacerbated by what?

- Pregnancy

What is the rate limiting step in glycolysis?

PFK-1

Fructose-6-Phosphate → Fructose-1,6-Bisphosphate: What enzyme catalyzes this rxn?

- Phosphofructokinase-1 (uses ATP)

- Commits glucose to glycolysis

Phosphofructokinase-1: Key Inhibitors and Inducers

Inhibitors (↓ Glycolysis):

- Citrate (from TCA cycle), ATP (MEANS THE CELL ALREADY HAS HIGH ENERGY)

Inducers (↑ Glycolysis):

- AMP, Fructose-2,6-Bisphosphate (MEANS THE CELL NEEDS MORE ENERGY)

What are the roles of PFK1 and Fructose-1,6-Bisphosphatase 1?

- PFK: F6P → F16BP (Glycolysis)

- Fructose-1,6-Bisphosphatase 1: F16BP → F6P (Gluconeogenesis)

The relative activity of the two enzymes will determine if the cell utilizes glycolysis or gluconeogenesis

How do Insulin/Glucagon control the rate of glycolysis/gluconeogenesis?

- Via Fructose-2,6-Bisphoshate

Fructose-2,6-Bisphoshate

- Activates PFK-1 → drives cell towards glycolysis

- Inhibits Fructose-1,6-BP1

Fructose-2,6-Bisphoshate: What does it do when its [c] is high or low?

- High → Glycolysis ON

- Low → Glycolysis OFF

Fructose-2,6-Bisphoshate: How is it synthesized?

- Fructose-6-Phosphate, one of the molecules its supposed to regulate

What is the role of PFK2/FBPase2?

- They control the conversion of F6P ↔ F26BP

How do Insulin and Glucagon affect Fructose-2,6-Bisphoshate? Its Result?

- Insulin: Dephosphorylates PFK2/F16BP → ↑ Fructose 2,6 BP → Activates Glycolysis

- Glucagon: Phosphorylates PFK2/F16BP → ↓ Fructose 2,6 BP → Activates Gluconeogenesis

Glycolysis: Splitting Stage

- 1x (6 carbon) molecule of Fructose-1,6-BP → 2x (3 carbon) molecules of Glyceraldehyde-3-Phosphate

Glycolysis: Energy Stage

- Starts with 2x Glyceraldehyde-3-Phosphate → ends in pyruvate

What Is Generated During the Energy Stage of Glycolysis?

- 4 ATP, 2 NADH (2x ATP and 1x NADH per GAP, and there are 2x GAP)

- 2 net ATP generated because 2x are used during investment stage

Phosphoenolpyruvate → Pyruvate: What enzyme catalyzes this rxn?

- Pyruvate Kinase

Pyruvate Kinase: Key Inhibitors/Inducers

- Inhibitors: ATP and Alanine

- Inducers: Fructose-1,6-BP (Feedforward activation)

What effect do Glucagon/Epinephrine have on Pyruvate Kinase? Its result?

- They phosphorylate it → inactivates PK

- Slows glycolysis and favors gluconeogenesis

Alanine: Where is it made/why? What do its levels in the body indicate?

- Where/Why: Skeletal muscles degrade proteins for energy → produces alanine → sent to blood/liver

- Indicates: A lack of glucose throughout the body

Why is Alanine an Inhibitor of Pyruvate Kinase?

- Indicates low [glucose] in body bc muscles are degrading proteins for energy

- When alanine goes to liver, it inhibits PK → slows down glycolysis; liver then converts alanine back into glucose via gluconeogenesis → sent back to muscles as glucose for energy

What is the end product of glycolysis? What can it generate?

- Product: Pyruvate

- Generates: Lactate or Acetyl-CoA (for TCA)

What happens when the TCA cycle is unavailable? (Ex. ↓ O2)

- Pyruvate is shunted to lactate

Lactate Dehydrogenase

- Converts pyruvate to lactate, uses NADH (regenerates NAD+)

When are levels of Lactate Dehydrogenase common?

- Hemolysis

- Myocardial Infraction

- Tumors

Level of NAD+ in cells

- They have a limited supply NAD+, needs to be regenerated in order to continue conducting glycolysis

How is NAD+ regenerated: O2 present vs O2 Absent?

- Present: NADH made in glycolysis can go to mitochondria → NAD+ regenerated in oxidative phosphorylation (that makes ATP)

- Absent: TCA/Oxidative phosphorylation are not available; pyruvate → lactate is important for regenerating NAD+

Lactic Acidosis: MOA

1. ↓ O2 → ↓ Pyruvate entry into TCA → Shunted to Lactic acid production to regenerate NAD+

2. ↑ Lactic acid → ↓ pH and HCO3

When is Lactic Acidosis seen clinically?

- Sepsis, bowel ischemia, seizures

Too much exercise can result in what?

1. Excessive exercise → NAD+ overcomsumed (TCA can't keep up)

2. Cells favor pyruvate → lactate

3. ↑ Lactate → ↓ pH in muscles → cramps

Pyruvate Kinase Deficiency: Inheritance; What age does it present at?

- Autosomal Recessive

- Usually presents in newborns

Pyruvate Kinase Deficiency: Which cells are most affected? Its result?

- Affected: RBCs bc/ they lack mitochondria

- Result: RBCs require PK for anaerobic metabolism. If deficient, → ↓ ATP → RBC membrane failure → Phagocytosis in spleen

Pyruvate Kinase Deficiency: Presentation

- Extravascular hemolysis: The destruction of red blood cells outside the blood vessels → Hemolytic Anemia

- Splenomegaly

Disease severity ranges based on enzyme activity

2,3 Bisphosphoglycerate

- Compound that lowers hemoglobin's affinity to oxygen, thereby freeing up oxygen for use by tissues; 2,3-BPG levels are higher in those acclimated to high altitudes

How much energy can a cell derive from a molecule of glucose using glycolysis?

ATP yield depends on whether cell has mitochondria and O2 present

Oxygen and Mitochondria Present

32-30 ATP

- 32: Malate-Aspartate Shuttle (Liver/Heart)

- 30: Glycerol-3-Phosphate Shuttle (Muscle)

Oxygen and Mitochondria Absent

2 ATP

What is the end product of glycolysis?

- Pyruvate

Why would Pyruvate be transported to the mitochondria?

- Can enter either TCA cycle (ATP) or Gluconeogenesis (Glucose)

How is Pyruvate transported to the mitochondria?

- Outer membrane: Voltage-gated porin complex

- Inner: Mitochondrial pyruvate carrier

What can Pyruvate encounter after moving into the mitochondria?

- Pyruvate Carboxylase: Shunts pyruvate back to gluconeogenesis

- Pyruvate Dehydrogenase Complex: Pyruvate → Acetyl-CoA → TCA

How does high [ATP] shunt pyruvate to gluconeogenesis?

1. High levels of ATP slow down TCA cycle

2. If TCA slows, Acetyl-CoA ↑↑↑

3. High [Acetyl-CoA] activates Pyruvate Carboxylase → shunts pyruvate to gluconeogenesis

Where does Pyruvate go when Acetyl-CoA levels are high/low?

- High: Gluconeogenesis

- Low: TCA for ATP

Pyruvate Dehydrogenase Complex

- Pyruvate Dehydrogenase (E1)

- Dihydrolipoly Transcetylase (E2)

- Dihydrolipoly Dehydrogenase (E3)

Pyruvate Dehydrogenase Complex: Cofactors

- NAD+

- FAD

- Coenzyme A (CoA)

- Thiamine

- Lipoic Acid

Pyruvate Dehydrogenase Complex: Role of E1

- Adds Thiamine-PP to Pyruvate → Adds carbon skeleton of pyruvate to thiamine pp

- Releases CO2

Pyruvate Dehydrogenase Complex: Role of E2

- Pyruvate:Thiamine is attached to lipoic acid

- Co-A is then added to this structure to generate Acetyl-CoA

Pyruvate Dehydrogenase Complex: Role of E3

- Uses NAD/FAD cofactors to keep lipoic acid in proper form, so E2 can use it to create Acetyl-CoA

Thiamine

- B1 vitamin necessary to use glucose