Gluconeogenesis

Question 1

What is gluconeogenesis

Answer 1

The formation of new glucose from non-carbohydrate sources

Question 2

What is the purpose of gluconeogenesis?

Answer 2

To provide glucose for export to other tissues when glycogen stores are exhausted ad when no dietary glucose is available

Question 3

Where does gluconeogenesis occur?

Answer 3

In the liver and kidneys (to a lesser extent)

Question 4

What hormones regulate gluconeogenesis?

Answer 4

• Glucagon
• Insulin

Question 5

What are some glucogenic precursors that can be utilized in gluconeogenesis?

Answer 5

• Pyruvate
• Lactate
• Amino acids (alanine)
• Glycerol

Question 6

Is gluconeogenesis glycolysis in reverse?

Answer 6

No!
- There are three irreversible steps in glycolysis (due to high -ΔG)
- These steps must be bypassed in gluconeogenesis

Question 7

What is the starting material for gluconeogenesis?

Answer 7

Any compound that can be converted to either pyruvate or oxaloacetate

Question 8

Of the 10 reactions of gluconeogenesis, how many are the reverse of glycolysis?

Answer 8

7

Question 9

What are the three reactions that must be bypassed in gluconeogenesis?

Answer 9

1. Conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase
2. The phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1)
3. Conversion of glucose to glucose 6-phosphate by hexokinase

Question 10

Describe the first bypass reaction in gluconeogenesis

Answer 10

1. Conversion of phosphoenolpyruvate (PEP) to pyruvate by pyruvate kinase

• Last step of glycolysis
• 2 steps

Step 1: Pyruvate → Oxaloacetate
- Occurs in the mitochondria
- Pyruvate is transported into the mitochondria via the malate shuttle
- Reaction is catalyzed by pyruvate carboxylase (2 active sites)
- Biotin (coenzyme) has long arms that can access both sites 1 and 2
- Site 1 - HCO3- uses energy from hydrolysis to form CO2 which is covalently added to biotin
- Site 2 - Biotin donates CO2 to pyruvate
Pyruvate + HCO3- + ATP → Oxaloacetate + ADP +Pi

Step 2: Oxaloacetate → Phosphoenolpyruvate (PEP)
- Occurs in the cytosol
- Utilize malate shuttle
- Catalyzed by PEP carboxykinase
Oxaloacetate + GTP ⇌ PEP + CO2 + GDP

Question 11

Why does the second part of the first bypass reaction need to utilize the malate shuttle?

Answer 11

• The second step in the first bypass reaction takes place in the cytosol BUT Oxaloacetate CANNOT freely transport across the mitochondria membrane
• Oxaloacetate gets converted to malate which can freely cross the mitochondria membrane by the malate transporter
• Malate gets reconverted to oxaloacetate in the cytosol of the mitochondria
• Catalyzed by malate dehydrogenase

Question 12

What is the significance of the malate shuttle?

Answer 12

• NADH is low in the cytosol compared to in the mitochondria
• The malate shuttle transports NADH from the mitochondria to the cytosol so that it can be used in subsequent gluconeogenesis reactions (conversion of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate)

Mitochondria: Oxaloacetate NADH + H+ ⇌ Malate + NAD+
Cytosol: Malate + NAD+ → Oxaloacetate + NADH + H+

Question 13

The first bypass reaction in which pyruvate → PEP described above is predominant when pyruvate is the glucogenic precursor BUT, lactate can also be used as a glucogenic precursor. How does the by pass reaction differ when lactate is the precursor?

Answer 13

• Lactate is able to bypass the malate shuttle
• Oxaloacetate is directly converted to PEP by using a mitochondrial version of PEP carboxykinase

Question 14

Describe the second and third bypass reaction in gluconeogenesis

Answer 14

2. The phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1)
3. Conversion of glucose 6-phosphate to glucose by hexokinase

These are both hydrolysis reactions that utilizes water

• Step 2 = catalyzed by fructose 1,6-bisphosphatase (FBPase-1)
  Fructose 1,6-phosphate + H2O → Fructose 6-phosphate +Pi
• Step 3 = catalyzed by Glucose 6-phosphatase
  Glucose 6-phosphate + H2O → glucose + Pi

Question 15

Gluconeogenesis is very energetically expensive. So, why does the cell carry out this reaction?

Answer 15

• There are many tissues (ex: brain) that rely heavily on glucose
• Irreversibility of gluconeogenesis ensures that gluconeogenesis and glycolysis do NOT occur simultaneously
  (Doing both at the same time is very wasteful!)
• Prevents the excretion (complete waste) of pyruvate
• Many molecules feed into gluconeogenesis

Question 16

How and why do we prevent gluconeogenesis and glycolysis from running at the same time?

Answer 16

PFK-1 and FBPase-1 reciprocally regulate glycolysis and gluconeogenesis to prevent both pathways from running at the same time (which is wasteful)
PFK-1 catalyzes the reaction that commits glucose 6-phosphate to glycolysis

Question 17

Describe the reciprocal regulation of PFK-1 and FBPase-1

Answer 17

• Both enzymes catalyze the reaction that converts Fructose 6-phosphate to Fructose 1,6-bisphosphate (reverse in gluconeogenesis)
• When ATP (or citrate) = high, PFK-1 is inhibited (ATP production is higher than consumption)
• ADP or AMP will relieve this inhibition (activate PFK-1) (ATP consumption is higher than ATP production)
• AMP inhibits FBPase-1

Question 18

What is responsible for the reciprocal regulation of PFK-1 and FBPase-1?

Answer 18

• Fructose 2,6-bisphosphate

Question 19

How does Fructose 2,6-bisphosphate allosterically regulate PFK-1 and FBPase-1

Answer 19

• PFK-1 = allosterically activated by Fructose 2,6-bisphosphate (reduced affinity for inhibitors citrate / ATP)
• FBPase-1 (second bypass step / gluconeogenesis equivalent of PFK-1) = allosterically inhibited by Fructose 2,6-bisphosphate

In other words….
- If Fructose 2,6-bisphosphate is present: PFK-1 activity is high and FBPase-1 activity = low

Question 20

Where does Fructose 2,6-bisphosphate come from?

Answer 20

Fructose 6-phosphate

Question 21

What catalyzes the reaction that creates fructose 6-phosphate?

Answer 21

PFK-2

Question 22

What regulates the breakdown of fructose 6-phosphate?

Answer 22

FBPase-2

Question 23

How are PFK-2 and FBPase-2 regulated?

Answer 23

• Phosphorylation
• PFK-2 and FBPase-2 are the same protein molecule. When the enzyme is phosphorylated FBPase-2 is active. When the enzyme is NOT phosphorylated PFK-2 is active.
• cAMP-dependent protein kinase phosphorylates enzyme activating FBPase-2
• Phospho-protein phosphatase removes phosphoryl group from enzyme activating PFK-2

Question 24

What role does glucagon play in the regulation of fructose 6-phosphate?

Answer 24

• Stimulates adenylyl cyclase to synthesize cAMP which in turn activates cAMP-dependent protein kinase
• Lowers cellular levels of fructose 2,6-bisphosphate (FBPase-2)
• Inhibits glycolysis and stimulates gluconeogenesis

Question 25

What role does insulin play in the regulation of fructose 6-phosphate?

Answer 25

- Stimulates the activity of phosphorus-protein phosphatase - Increases cellular levels of fructose 2,6-bisphosphate (PFK-2) - Inhibits gluconeogenosis

Question 26

How else can PFK-2 and FBPase-2 be regulated?

Answer 26

- Xylulose 5-phosphate - Dephosphorylates PFK-2 / FBPase-2 enzyme activating PFK-2 / inhibiting FBPase-2 inhibiting gluconeogenesis and stimulating glycolysis

Question 27

How does the spatial separation of glycolysis and gluconeogenesis prevent both processes from occurring at the same time?

Answer 27

- Glucose 6-phosphatase (last bypass reaction) is exclusively found on the ER membrane of the liver and kidneys - The active site of glucose 6-phosphatase faces the ER lumen - Glucose 6-phosphate = converted to glucose in the ER lumen - Glucose is then transported to the cytosol (very briefly) before it is transported back out of the cytosol and into the bloodstream *Because glucose is NOT in the cytosol for a long period of time it cannot enter glycolysis*