Gluconeogenesis Lecture Sep 19

Question 1

WHat are the three key regulatory steps of glycolysis?

Answer 1

glucokinase (glucose to glucose 6 phosphate; inhibited by product inhibition)

PFK-1 (fructose 6 phosphate to fructose 1,6, bisphosphate with allosteric regulation: inhibition by ATP and citrate; activation by AMP and fructose 2,6, bisphosphate)

Pyruvate kinase (PEP to pyruvate; in the liver inhibited by phosphorylation from PKA)

Question 2

WHat are the key regulatory steps of gluconeogenesis?

How do they relate to the key regulatory steps of glycolysis?

Answer 2

THey are the same as the key regulatory steps in glycolysis! Only with different enzymes….

Glucose 6 phosphatase (turning glucose 6 phosphate to glucose - only present in the liver)

Fructose 1,6, bisphosphatase (turning fructose 1,6 bisphosphate into fructose 6 phosphate; transcription increase by cortisol, inhibited by AMO and F2,6,P)

Phosphoenolpyruvate carboxykinase (transcriptionally regulated by cAMP and cortisol)

Question 3

After the glucose from a recent meal is consumed, does the liver great glucose first from gluconeogenesis or glycogenolysis?

Answer 3

First from glycogenolysis

Then after glycogen stores are depleted, from gluconeogenesis

Question 4

What are the three sources of carbon for gluconeogenesis?

What is the energy soure for gluconeogenesis?

Answer 4

The three sources of carobn are from:

anaerobic glycolysis in the form of lactate (always happening in RBCs),

degradation of muscle proteins in the form of AAs - mostly alanine, and from

triacylglycerol lipolysis supply of glycerol

The energy supply comes from the ATP produced by beta oxidation of fatty acids

Question 5

What enzyme and cofactor will allow lactate to be used in gluconeogenesis?

Answer 5

Lactate dehydrogenase will interconvert lactate to pyruvate (with NAD as a cofactor)

the pyruvate can then be a substrate for PEP CK and enter gluconeogenesis

Question 6

WHat enzyme will allow alanine to be used for gluconeogenesis?

Answer 6

alanine aminotransferase will remove the amine from alanine, thus converting it to pyruvate, which can be a substrate for PEP CK in gluconeogenesis.

Question 7

WHat enzymes are necessary for glycerol to be used in gluconeogenesis? At what level does it enter?

Answer 7

Glycerol is released from the lipolysis of TAGs

Glycerol kinase will use ATP to convert glycerol to glycerol 3 phosphate.

Glycerol 3 phosphate dehydrogenase will use NAD+ to convert glycerol 3 phosphate to dihydroxyacetone phosphate

DHAP can then be a substrate for aldolase to synthesize fructose 6 phosphate in gluconeogenesis

Question 8

Besides using different enzymes, how is the conversion of pyruvate to PEP in gluconeogenesis different from the conversion of PEP to pyruvate in glycolysis?

Answer 8

In glycolysis, the conversion happens in one step.

In gluconeogenesis it requires multiple reactions.

1. Pyruvate is carboxylated to oxaloacetate in the mitochondria.
2. The oxaloacetate is transported to the cytosol in the form of eithe rmalate or aspartate
3. THen its converted back to oxaloacetate in the cytosol
4. PEP CK then converts the cytosolic oxaloacetate to PEP.

Question 9

Which gluconeogenic substrate will enter gluconeogenesis bypassing the PEP CK regulatory step?

Answer 9

Glycerol, which doesn’t enter at the level of pyruvate. It enters as DHAP.

Question 10

In the conversion of pyruvate to PEP in gluconeogensis, what enzyme first converts the pyruvate to oxaloacetate? What’s the cofactor? Where does it happen?

Answer 10

In the mitochondrial matrix, pyruvate is converted to oxaloacetate by pyruvate carbosylase using biotin as a cofactor (addition of a CO2).

Question 11

WHat determines whether the oxaloacetate will be transported out of the mitochondria as aspartate or malate?

Answer 11

The balance between cytosolic and mitohcondrial pools of NADH.

The conversion of oxaloacetate to malate requires NADH, but the conversion of oxaloacetate to aspartate does not.

SO…

If NADH is higher in the mitochondria, it will transport oxaloacetate as malate.

If NADH is higher in the cytosol, it will transport the oxaloacetate as aspartate.

Question 12

In the conversion of pyruvate to PEP,

once the oxaloacetate is transported to the cytosol, what enzyme converts it to PEP?

WHere does the energy for this come from?

Answer 12

PEP CK does the reaction with energy from GTP hydrolysis, removing CO2.

Question 13

How is the PEP CK step regulated?

Answer 13

It’s regulated through transcription. When the tissue wants to make glucose, it transcriptionally upregulates PEP CK through cAMP and cortisol:

1. When glucagon binds its receptor in the fasted state, the receptor activate adenyl cyclase and increases cAMP. cAMP then activates PKA, which phosphorylates the carbohydrate response element binding protein (CREB) and activates it. Activated CREB will then bind to the response element on the DNA and activate transcription of the PEP CK gene.
2. Cortisol will diffuse across the plasma membrane and bind its receptor, which is a ligand activated transcription factor. The activated transcription activator will bind the glucocorticoid response element on the DNA and initiate PEP CK gene transcription as well

Question 14

What does the promoter of the PEP CK gene contain?

Answer 14

1. insulin response element (exerts an inhibitory force when bound)
2. glucocorticoid response element (bound by the cortisol receptor when activated)
3. thyroid response element
4. two cAMP response elements - CRE1 and 2 (bound by the activated CREB)

Question 15

How does metformin work to control insulin resistant diabetes?

Answer 15

Metformin activates LKB1, which phosphorylates and activates AMPK. AMPK phosphorylates TORC2 and prevents its nuclear localization.

If TORC2 gets into the nucleus, it binds a CREB and they then bind the DNA to increase expression of PCG1alpha

PGC1alpha expression leaves to the increased gluconeogenic gene expression which enhances gluconeogensis

By phosphorylating TORC2, this doesn’t happen

Question 16

How does the body prevent futile cycling during gluconeogenesis in terms of the PEP and pyruvate conversion?

Answer 16

In the fasted state:

1. pyruvate kinase is inactivated by PKA
2. PDH is inactivated by PDH kinase, which is activated by acetyl CoA (of which there is a bunch in the fasted state because you have hormone sensitive lipase active)
3. PEP CK is activated by PKA and corisol

Thus, the pyruvate carboylase reaction is favored and you get conversion of pyruvate to oxaloacetate and then PEP.

Question 17

What enzyme is used for the fructose 6-P to fructose 1,6, bisP reaction in glycolysis?

What enzyme is used for the fructose 1-6 bisP to fructose 6-P reaction in gluconeogenesis?

Answer 17

glycolysis: PFK-1

(inhibited by ATP and citrate; activated by AMP and fructose 2,6, bisphosphate)

Gluconeogenesis: Fructose 1,6 bisphosphatase

(inhibited by AMP and 2,6, bisphosphate)

Question 18

How does the body avoid futile cycline at the frustose 1,6, bisphosphate to fructose 6 phosphate step in gluconeogenesis?

Answer 18

1. During glycolysis, fructose 1,6, bisphosphatase is inhibited by fructose 2,6-bisphosphate (which is in high levels through the PFK-2 reaction on high levels of fructose 6 phosphate in the fed state)
2. During gluconeogenesis, PFK-1 is shut off because there is little fructose 2,6, bisphosphate to activate it in the fasted state.

So F2,6-BP is in high concentrations in the fed state to activate PFK-1 and inhibit F1,6-BPase

Question 19

The first step in glycolysis/last step in gluconeogenesis is also a potential area for futile cycling. How does the body avoid it?

Answer 19

Futile cycling is prevented in the conversion of glucose 6-phosphate to glucose by the different Km values of glucokinase and hexokinase.

Gluconeogenesis happens in the liver, where glucokinase is used - everything else uses hexokinase. Because hexokinase has a lower Km than glucokinase, the kinetics favor glucose exiting hte liver and the production of glucose 6 phosphate in other tissues.

In other words, the glucose is pulled out of the liver cell due t othe higher affinity of hexokinase. Thus, the glucose isn’t even there to be futily cycled back to glucose 6 phosphate.

Question 20

Besides the higher affinity for glucose by hexokinase than glucoskinase, what else promotes the transport of glucose out of the liver to peripheral tissues?

Answer 20

THe relative affinities of the GLUT transports. THe liver uses GLUT 2 which has the lowest affinity, while the peripheral (and all cells basically) have Glut 1 and Glut 3, which have high affinity for glucose.

Question 21

WHat are the three diseases related to gluconeogenesis?

Answer 21

GLucose 6 phosphatase deficiency (GSD1 - von Gierke’s disease). This is an inability to break down glycogen and an inability to undergo gluconeogenesis. It results in fasting hypoglycemia, lactic acidosis, and an enlarged liver

Fructose 1,6-bisphosphatase deficiency - this is a rare autosomal recessive disorder with episodic hypoglycemia when liver glycogen is depleted. THis person is unable to do gluconeogenesis period (regardless of whether the carbon is coming from lactate, alanine or glycerol)

PEP CK deficiency - this may not even exist really. Someone with this disorder would have fasting hypoglycemia. BUt this person could do SOME gluconeogenesis if glycerol was the carbon donor, because glycerol bypasses the PEP CK step.