Gluconeogenesis

Question 1

Differentiate glycolysis and gluconeogenesis

Answer 1

Glycolysis starts with glucose and forms pyruvate.

Gluconeogenesis starts with pyruvate and forms glucose

Question 2

What is the purpose of gluconeogenesis?

Answer 2

1. Provision of blood glucose for cells that need glucose for synthesis
   of ATP and other specific molecules like for examples:
2. Synthesis of glucose from non-carbohydrates. Mainly from amino acids, lactate or glycerol which are provided via the blood under hormonal control.
3. Sole provider for blood glucose once hepatic glycogen is depleted.
   Essential for survival during starvation

Question 3

Where does gluconeogenesis occur?

Answer 3

Liver and kidneys perform gluconeognesis in well-oxygenated
hepatic periportal cells and renal cortex cells

80%-liver

20%-kidney

Question 4

When is gluconeogenesis performed?

Answer 4

Gluconeogenesis takes place at low blood glucose levels during fasting and flight & fight situations (stress).

Glucagon
stimulates gluconeogenesis and inhibits hepatic glycolysis.

This hormonal regulation ensures that glycolysis and gluconeogenesis do not take place at the same time in the same hepatic cell

Question 5

Gluconeogenesis is not…

Answer 5

Gluconeogenesis is not a reversal of glycolysis

The irreversible steps of glycolysis are bypassed
by irreversible steps of gluconeogenesis

Question 6

Summarize hepatic metabolism at low blood glucose

Answer 6

Glycogen degradation and gluconeogenesissupply glucose 6-P to supply free glucose

Gluconeogenesisis stimulated by alanine, glutamine, lactate, and glycerol

Free fatty acids are converted to B -oxidation of fatty acids-provision of ATP and allosteric regulations by NADH and acetyl CoA

Question 7

Fatty acid degradation is needed for gluconeogenesis but why are the carbons of acetyl CoA not used to form glucose?

Answer 7

• Acetyl CoA formed during fasting by fatty acid degradation cannot be used to form pyruvate, as the PDH complex is irreversible.
• Acetyl CoA is normally substrate for citrate synthase, but during fasting, the hepatic TCA cycle is inhibited by NADH at isocitrate DH. This NADH is formed in fatty acid degradation.
• Acetyl CoA even if used in the TCA cycle does not lead to an additional molecule that could be used for gluconeogenesis. Acetyl CoA adds two carbons but two carbons are lost in two decarboxylation steps

Question 8

What is the glucose-alanine cycle?

Answer 8

The glucose-alanine cycle describes
hepatic gluconeogenesis using alanine which was released by muscle.

The formed glucose is released by the liver into the blood and can be taken up by muscle and other tissues.

Question 9

What is the Cori-cycle?

Answer 9

The Cori-cycle describes
hepatic gluconeogenesis using lactate which was released by muscle.

Lactate is formed during anaerobic glycolysis in muscle.

The formed glucose is released by the liver into the blood and can be taken up by muscle for anaerobic glycolysis.

note: The release of lactate from RBC or
other cells performing anaerobic glycolysis is sometimes included in the Cori-cycle.

Question 10

How is pyruvate for gluconeogenesis mainly formed?

Answer 10

A. Lactate is oxidized to pyruvate by lactate dehydrogenase

B. Alanine is transaminated to pyruvate by alanine aminotransferase

Question 11

What happens to pyruvate?

How can it be used for gluconeogenesis? How is PEP formed?

Answer 11

There is no single enzyme that bypasses
this irreversible step of pyruvate kinase of glycolysis.

Two enzymes are needed in gluconeogenesis:

1. Pyruvate carboxylase (mitochondria)
2. PEP carboxykinase (cytosol)

Question 12

What happens to pyruvate after entering the mitochondria?

Answer 12

Pyruvate enters mitochondria and is carboxylated to oxaloacetate

Acetyl CoA formed during fasting by b-oxidation of fatty acids inhibits PDH complex and activates pyruvate carboxylase: this shifts pyruvate to gluconeogenesis

Question 13

What is the significance of PEP carboxylase and PEP carboxykinase?

Answer 13

• Pyruvate carboxylase is found in mitochondria and uses pyruvate, carbon dioxide and ATP for formation of oxaloacetate. The vitamin biotin is covalently bound to the enzyme and interacts with carbon dioxide.
• Pyruvate carboxylase need absolute activation by the allosteric activator acetyl CoA.
• During gluconeogenesis acetyl CoA is formed by fatty acid degradation in mitochondria.
• If a patient cannot perform fatty acid degradation, that result also in reduced gluconeogenesis.
• The carbon dioxide (shown in green circle) added by pyruvate carboxylase forming oxaloacetate will be released in the PEP carboxykinase reaction generating energy for that reaction.
• Oxaloacetate formed in mitochondria cannot pass through the inner mitochondrial membrane. Malate is formed and enters the cytosol using a malate transporter.
• In the cytosol: malate and NAD+ are used to form cytosolic oxaloacetate which is a substrate for cytosolic PEP carboxykinase.
• PEP carboxykinase uses GTP and forms PEP, GDP and carbon dioxide.
• Cortisol induces PEP carboxykinase and is needed for long term gluconeogenesis.
• PEP joins gluconeogenesis

Question 14

Why can gluconeogenesis not use carbons from Acetyl CoA?

Answer 14

Glucogenic amino acid degradation generates pyruvate or it provides additional intermediates of the TCA cycle that can be used for gluconeogenesis.

• AcetylCoA(fattyaciddegradation)cannotbeusedinthefollowing:

a. The PDH reaction is irreversible and pyruvate cannot be formed.
b. The hepatic TCA cycle during fasting is inhibited by NADH at isocitrate DH.
c. The TCA cycle cannot lead to an additional molecule of oxaloacetate as although
acetyl CoA adds two carbons, two carbons are lost as CO2 in the cycle.

• Acetyl CoA from b-oxidation is however needed as an absolute
allosteric activator for pyruvate carboxylas

Question 15

What substrates are provided for gluconeogenesis?

Answer 15

Substrates for gluconeogenesis provided by the blood:

Alanine, glutamine, lactate and glycero

Question 16

How are gluconeogenic amino acids used for gluconeogenesis?

Answer 16

Glucogenic amino acid degradation generates pyruvate or it provides
additional intermediates of the TCA cycle that can be used for gluconeogenesis.

Mainly alanine and glutamine are provided by other tissues and transported in blood to the liver.

Mainly alanine from the blood used for pyruvate

a-ketoglutarate-mainly glutamine from the blood

Question 17

Which cells release free fatty acids and glycerol into the blood at low blood glucose level?

Answer 17

Fat cells degrade their stored TAGs only at low blood glucose levelss

This leads to:
-Release of free fatty acids for energy metabolism
of other cells
-Release of free glycerol for gluconeogenesis

The released free fatty acids from fat cells are taken up glucose levels
from the blood and used for b-oxidation in the liver and many other cells.
Glycerol is used for gluconeogenesis

Question 18

What is the significance of free glycerol being released?

Answer 18

Free glycerol is released from fat cells at low blood glucose level and is used for gluconeogenesis

Low blood glucose leads fat cells to TAG degradation.
Free fatty acids and free glycerol are released into the blood.

Glycerol is then turned to glycerol-3-P and used for gluconeogenesis

Question 19

What is the most important short-term regukated enzyme of gluconeogenesis?

Answer 19

Fructose 1,6-bisphosphatase is the most important short-term regulated enzyme of gluconeogenesis

Inhibited by fructose 2,6 bisphosphate and AMP

Question 20

How and when is fructose 2,6-bisphosphate (F2,6-bisP) formed and what is its function in hepatocytes?

Answer 20

F2,6-bisP is formed by the hepatic bifunctional enzyme
at high blood insulin/glucagon ratio and it is needed for allosteric reciprocal regulation in the liver.

Some molecules of F2,6-bisP
Activate PFK-1 and enable hepatic glycolysis at normal ATP levels whereas other molecules Inhibit fructose 1,6-bisphosphatase and prevent gluconeogenesis.

High blood glucose levels activate hepatic glycolysis and inhibit gluconeogenesis

Question 21

What does the bifunctional(F2,6 BP) enzyme form?

Answer 21

The bifunctional enzyme does not only form F2,6-bisP from fructose 6-P but it also degrades it to fructose 6-P.

There is only one gene and one protein for this enzyme which is unusual as it has two counteracting irreversible enzyme activities.

The bifunctional enzyme forms F2,6-bisP with its PFK-2 activity and degrades F2,6-bisP with its BPase-2 activity.

This is why the bifunctional enzyme is folded in a way that only one activity can be performed at a time, either PFK-2 or BPase-2.

Question 22

How are bifunctional enzyme activities switched over in the liver?

Answer 22

The hepatic bifunctional enzyme can be phosphorylated by PKA.

Glucagon leads to the phosphorylated bifunctional enzyme which makes a conformational shift that leads to an inactivate PFK-2 and an active BPase-2. F2,6-bisP is degraded which promotes gluconeogenesis.

Insulin leads to the dephosphorylated bifunctional enzyme that forms fructose 2,6-bisP and promotes glycolysis (PFK-2 is active)

Question 23

What is the last enzyme in the gluconeogenesis pathway? Where is it found?

Answer 23

glucose-6 phosphatase

Found in the renal cortex and the liver

Glucose 6-phosphatase is bound in the ER membrane and acts toward the ER lumen.

Glucose 6-P is transported from cytosol into the ER lumen and is cleaved there to free glucose and inorganic phosphate which are transported into cytos

Question 24

What is the purpose of Glucose 6-phosphatase?

Answer 24

Glucose 6-phosphatase forms free glucose from gluconeogenesis and from glycogen degradation.

Both pathways, glycogen degradation and gluconeogenesis take place in the liver at low blood glucose levels at the same time favored by glucagon.

Deficiency of glucose 6-phosphatase strongly reduces the release of glucose
into the blood and leads to severe hypoglycemia and lactic acidosi

Question 25

Why is B-oxidation needed for gluconeogenesis ?

Answer 25

Fatty acid degradation in mitochondria and oxidative phosphorylation provide the ATP for hepatic functions during low blood glucose level where the liver forms but not uses glucose.

Gluconeogenesis starting with 2 pyruvates needs 4 ATP, 2 GTP and 2 NADH.

b-oxidation provides also allosteric regulators that allow the switch from glycolysis to gluconeogenesis

Question 26

When do gluconeogenesis and mitochondrial B-oxidation of fatty acids ?

Answer 26

Gluconeogenesis and mitochondrial b-oxidation of fatty acids have to take place at the same time during fasting in hepatocytes.

Pyruvate in this case is used fir both gluconeogenesis and PDH complex is inhibited

B-oxidation of fatty acids is absolutely needed. This pathway provides ATP and generates NADH and acetyl CoA for allosteric regulations

Question 27

Describe the release of free fatty acids and free glycerol into the blood

Answer 27

Low serum insulin and high levels of epinephrine and cortisol lead to activation of hormone-sensitive lipase inside of fat cells.

The release of free fatty acids into the blood provides other cells with substrates for energy metabolism.

In hepatocytes fatty acid degradation generates energy and acetyl CoA and NADH for allosteric regulations needed for gluconeogenesis

Question 28

Describe the uptake of free fatty acids from the blood and B-oxidation in mitochondria in all cells for energy metabolism

Answer 28

One spiral of the fatty acid b-oxidation generates the following:

• FADH2
• NADH
• Fatty acyl CoA (2 C shorter)
• Acetyl CoA

Only in the liver and kidney, acetyl CoA and NADH are needed for allosteric regulations that activate gluconeogenesis and inhibit glycolysis and PDH and reduce the TCA cycle.