Carbohydrate Metabolism: Gluconeogenesis

Question 1

Gluconeogenesis is also known as carbohydrates __________.

Answer 1

anabolism

Question 2

What is gluconeogenesis? Where does it occur?

Answer 2

Gluconeogenesis is the biosynthesis of glucose from non-carbohydrate precursors. Live a reverse glycolysis but the three irreversible reactions (1, 3, and 10 of glycolysis) must be bypassed.

Gluconeogenesis take places in the LIVER (main) and kidney (~20% of glucose)

Question 3

Brain requires _____ grams/day of glucose, out of the _____ g needed by the entire body,

Glycogen reserves can provide ____ g of it and glucose in body fluids is ____g.

When glucose is depleted (fasting/prolonged exercise), glucose must be synthesized from other sources: ____________.

Answer 3

120; 160

190; 20

Gluconeogenesis

Question 4

While glycolysis mainly occurs in the _____ and the _______, gluconeogenesis mainly occurs in the ______.

Answer 4

muscles; brain; liver

*glycolysis takes place in liver as well

Question 5

What are the five gluconeogenesis substrates/precursors; how important are they + what is their origin?

Answer 5

1) Pyruvate and lactate (1/3 - RBC and muscles)

2) Alanine (1/3 - muscles) - form of lactate

3) Glycerol (1/12 - adipose tissue, food lipid catabolism + lipoproteins)

4) All amino acids except leucine and lysine (1/8 - food/tissue proteins)

5) Propionyl-CoA (1/8 - oxidation of odd number fatty acid)`

Question 6

(T/F) Anything from the Kreb’s cycle can become glucose.

Answer 6

True!

Question 7

Match the 3 irreversible reactions in glycolysis to the enzymes involved in glycolysis and gluconeogenesis:

In glycolysis:
1) Reaction 1
2) Reaction 3
3) Reaction 10

A) Phosphofructokinase
B) Phosphoenolpyruvate carboxykinase
C) Glucose-6-phosphatase
D) Hexokinase
E) Fructo-1,6-bisphosphatase
F) Pyruvate kinase
G) Pyruvate carboxylase

Answer 7

In glycolysis
1) Reaction 1: Hexokinase
2) Reaction 3: Phosphofructokinase
3) Reaction 10: Pyruvate kinase

In gluconeogenesis
1) Reaction 1: Glucose-6-phosphatase
2) Reaction 3: Fructo-1,6-bisphosphatase
3) Reaction 4: Pyruvate carboxylase and Phosphoenolpyruvate carboxykinase

Question 8

How many gluconeogenesis reactions are necessary to bypass glycolysis reaction 10?

Answer 8

Two!

Therefore, gluconeogenesis has 11 reactions while glycolysis has 10!

Question 9

Match the first two reactions of gluconeogenesis to:

1) Reaction 1
2) Reaction 2

A) Phosphorylation and decarboxylation of oxaloacetate into phosphoenolpyruvate

B) Carboxylation of pyruvate into oxaloacetate

Answer 9

Reaction 1: Carboxylation of pyruvate into oxaloacetate

Reaction 2: Phosphorylation and decarboxylation of oxaloacetate into phosphoenolpyruvate (PEP)

Question 10

Why was a carboxyl added in the first reaction of step 1 of gluconeogenesis just to be removed in the second reaction?

Answer 10

The CO2 provided electrons necessary for the P-O bond that formed in the second reaction.

The addition of the carboxyl made the substrate better for the following reaction.

Question 11

Match the first two reactions of gluconeogenesis:

1) Reaction 1
2) Reaction 2

A) catalyzed by PHOSPHOENOLPYRUVATE CARBOXYKINASE (PEPCK) (in the cytosol)

B) catalyzed by PYRUVATE CARBOXYLASE (only found in the mitochondrial matrix, where this reaction takes place)

Answer 11

Reaction 1: catalyzed by PYRUVATE CARBOXYLASE (only found in the mitochondrial matrix, where this reaction takes place). An ATP is used.

Reaction 2: catalyzed by PHOSPHOENOLPYRUVATE CARBOXYKINASE (PEPCK) (in the cytosol). A GTP is used.

Question 12

(T/F) Pyruvate carboxylase is also used to convert pyruvate into oxaloacetate to REPLENISH THE intermediates of the TCA cycle, which takes place in the mitochondrial matrix (anaploresis).

Answer 12

True!

Question 13

Pyruvate enters the mitochondira via the _______ ______ ______ (what is it made of?).

This transport mechanism is also used for the conversion of _________ into _________.

Answer 13

Mitochondrial Pyruvate Carrier (MPC); heterodimer of MPC1 and MPC2

Pyurvate; Acetyl-CoA (TCA cycle subsrate)

Question 14

1) To exit the mitochondira, oxaloacetate must be ______ to ______ by the mitochondrial ______ ___________.

2) Once in the cytosol, ________ is _______ to oxaloacetate by the cytosolic ___________ __________.

3) Why isn’t there a transporter for oxaloacetate?

Answer 14

1) Reduced (gain electrons); Malate; Malate Dehydrogenase (MDH2)

2) Malate; Oxidized (lose electrons); Malate Dehydrogenase (MDH1)

3) No transporter for oxaloacetate because if there was, the TCA cycle will always pump it outside. But we want it inside the mitochondira.

Question 15

1) Which molecule is used to reduce oxaloacetate into malate so it can leave the mitochondria?

2) Which molecule is used to oxidize malate into oxaloacetate in the cytosol?

3) What shuttle is used to export malate into the cytoplasm?

Answer 15

1) NADH (making NAD+)

2) NAD+ (making NADH)

3) The malate-aspartate shuttle. Mainly, the MALATE α-KETOGLUTARATE TRANSPORTER.

Question 16

(T/F) One important aspect of the first reaction of gluconeogenesis is the fact that reducing equivalents (stored on NAD+) are transported from the mitochondria to the cytosol.

These reducing equivalents (NADH) will be used later by the GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH).

Answer 16

True!

There is not a lot of NADH in the cytosol. This reaction takes two electrons from mitochondria to the cytosol.

Question 17

What is the energy profile of the first bypass?

Answer 17

Pyruvate + ATP + GTP –> Phosphoenolpyruvate + ADP + GDP + Pi + H+

Delta G in intracellular conditions is -25kJ/mol (exergonic).

Two high energy phosphates must be invested for the synthesis of one phosphoenolpyruvate.

Since two phosphoenolpyruvates are needed to produce one molecule of glucose, TWO ATPs and TWO GDPs are used in the first bypass!

Question 18

What is the alternative path for the first bypass? When is it used?

Answer 18

The alternative path for the first bypass is mainly used when LACTATE is the substrate.

Lactate turns into pyruvate in the cytosol, then pyruvate enters the mitochondria which turns into oxaloacetate and then into PEP using mitochondrial PEP carboxykinase.

There is a transporter for PEP

Question 19

What is the alternative path for the first bypass? When is it used?

Answer 19

The alternative path for the first bypass is mainly used when LACTATE is the substrate.

Lactate turns into pyruvate in the cytosol, then pyruvate enters the mitochondria which turns into oxaloacetate and then directly into PEP using mitochondrial PEP carboxykinase.

Cytosolic NADH is generated by lactate dehydrogenase when lactate is turned into pyruvate. Therefore, there is no need to carry reducing equivalents (NADH) outside of the mitochondria, so no reason for malate!

Question 20

Reaction 3 of glycolysis is reaction _____ of gluconeogenesis which is catalyzed by ___________ in glycolysis.

Answer 20

9; PHOSPHOFRUCTOKINASE

Question 21

What is reaction 9 of gluconeogenesis? What is it catalyzed by?

Answer 21

HYDROLYSIS of fructose-1,6-bisphosphate into fructose-6-phosphate.

Reaction is catalyzed by the FRUCTOSE-1,6-BISPHOSPHATASE (FBPase1)

Question 22

(T/F) The fructose-1,6-bisphosphatASE was shown to be a key enzyme in regulating the metabolic rate of hibernating animals, the enzyme activity is temperature sensitive: it is less active when it is cold and therefore inhibits gluconeogenesis.

Answer 22

True!

Question 23

What is reaction 11 of gluconeogenesis? What is it catalyzed by?

Answer 23

It is the bypass glycolysis reaction 1 done by hexokinase.

It is the HYDROLYSIS of glucose-6-phosphate into glucose. Reaction is catalyzed by the GLUCOSE-6-PHOSPHATASE.

Question 24

Match the 11 reactions of gluconeogenesis:

1) Reaction 1
2) Reaction 2
3) Reaction 3
4) Reaction 4
5) Reaction 5
6) Reaction 6
7) Reaction 7
8) Reaction 8
9) Reaction 9
10) Reaction 10
11) Reaction 11

A) (2) Oxaloacetate becomes (2) Phosphoenolpyruvate (PEP) by PEP CARBOXYKINASE

B) (2) 1,3-bisphosphoglycerate (BPG) becomes (2) glyceraldehyde 3-phosphate (GAP) by GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH)

C) Fructose 1,6-bisphosphate (FBP) becomes Fructose-6-bisphosphate (F6P) by FRUCTOSE-1,6-BISPHOSPHATASE (FBPase1)

D) Glucose-6-phosphate (G6P) becomes Glucose by GLUCOSE-6-PHOSPHATASE!

E) (2) 2-phosphoglycerate (2PG) becomes (2) 3-phosphoglycerate (3PG) by PHOSPHOGLYCERATE MUTASE

F) dihydroxyacetone phosphate (DHAP) and (1) glyceraldehyde 3-phosphate (GAP) becomes (1) fructose 1,6-bisphosphate (FBP) by ALDOLASE

G) Fructose-6-bisphosphate (F6P) becomes Glucose-6-phosphate (G6P) by GLUCOSE-6-PHOSPHATE ISOMERASE

H) (1) glyceraldehyde 3-phosphate (GAP) becomes (1) dihydroxyacetone phosphate (DHAP) and by TRIOSE PHOSPHATE ISOMERASE (TPI)

I) (2) Pyruvate becomes (2) Oxaloacetate by PYRUVATE CARBOXYLASE

J) (2) 3-phosphoglycerate (3PG) becomes (2) 1,3-bisphosphoglycerate (BPG) by PHOSPHOGLYCERATE KINASE (PGK)

K) (2) PEP becomes (2) 2-phosphoglycerate (2PG) by ENOLASE

Answer 24

Reaction 1*: (2) Pyruvate becomes (2) Oxaloacetate by PYRUVATE CARBOXYLASE

Eeaction 2*: (2) Oxaloacetate becomes (2) Phosphoenolpyruvate (PEP) by PEP CARBOXYKINASE

Reaction 3: (2) PEP becomes (2) 2-phosphoglycerate (2PG) by ENOLASE

Reaction 4: (2) 2-phosphoglycerate (2PG) becomes (2) 3-phosphoglycerate (3PG) by PHOSPHOGLYCERATE MUTASE

Reaction 5: (2) 3-phosphoglycerate (3PG) becomes (2) 1,3-bisphosphoglycerate (BPG) by PHOSPHOGLYCERATE KINASE (PGK)

Reaction 6: (2) 1,3-bisphosphoglycerate (BPG) becomes (2) glyceraldehyde 3-phosphate (GAP) by GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH)

Reaction 7: (1) glyceraldehyde 3-phosphate (GAP) becomes (1) dihydroxyacetone phosphate (DHAP) and by TRIOSE PHOSPHATE ISOMERASE (TPI)

Reaction 8: (1) dihydroxyacetone phosphate (DHAP) and (1) glyceraldehyde 3-phosphate (GAP) becomes (1) fructose 1,6-bisphosphate (FBP) by ALDOLASE

Reaction 9*: Fructose 1,6-bisphosphate (FBP) becomes Fructose-6-bisphosphate (F6P) by FRUCTOSE-1,6-BISPHOSPHATASE (FBPase1)

Reaction 10: Fructose-6-bisphosphate (F6P) becomes Glucose-6-phosphate (G6P) by GLUCOSE-6-PHOSPHATE ISOMERASE

Reaction 11*: Glucose-6-phosphate (G6P) becomes Glucose by GLUCOSE-6-PHOSPHATASE!

Question 25

Gluconeogenesis uses __ pyruvate, __ ATP,
__ GTP, __ NADH, __ H+, and __H2O

to produce

__ glucose, __ ADP, __GDP, __ NAD+, and __Pi

Answer 25

2; 4; 2; 2; 2; 4

1; 4; 2; 2; 6

Question 26

Why can’t glycolysis operate in reverse?

Answer 26

Glycolysis can not operate in reverse because the energy cost is too much. The ΔG°’ is ~+70kJ/mol.

Therefore, by doing bypass of the 3 irreversible reactions of glycolysis in gluconeogenesis, ΔG°’ is brought down to -42.7 kJ/mol.

Question 27

Despite gluconeogenesis being costly, why is it physiologically necessary?

Answer 27

Brain, nervous system and RBCs generate ATP mostly from glucose.

When glycogen stores are depleted (lots being used), we need to get glucose from somewhere.

This can occur in between meals but is mostly during the night. It is done in more extreme conditions like starvation or vigorous exercise.

*it is however the last resort

Question 28

(T/F) Gluconeogenesis can also occur from other substrates than pyruvate. Some of them include lactate, glycerol, and amino acids.

Answer 28

True!

Question 29

(T/F) During aerobic glycolysis, muscle and erythrocytes produce lactate, which travels to the liver and gets converted back into glucose. Then, it returns to the blood and becomes available for muscle cells and erythrocytes.

Answer 29

False!

During ANAEROBIC glycolysis, muscle and erythrocytes produce lactate, which travels to the liver and gets converted back into glucose. Then, it returns to the blood and becomes available for muscle cells and erythrocytes.

Question 30

What is the cori cycle?

Answer 30

Cori cycle is when lactate is re-oxidized to pyruvate by the liver LACTATE DEHYDROGENASE (LDH).

This produces cytosolic NADH, so it follows the PEP alternative pathway where pyruvate enters the mitochondria and becomes oxaloacetate and then into PEP using mitochondrial PEP carboxykinase.

Then PEP leaves the mitochondria and becomes glucose through gluconeogenesis.

Question 31

Explain how gluconeogenesis occurs using glycerol:

Answer 31

When carbohydrates (glucose + glycogen) gets depleted, fatty acids become the principal metabolic fuel to fulfill the energy demand.

CATABOLSIM of triacylglycerides leads to the production of GLYCEROL and three free fatty acids.

The fatty acids get used as fuel for TCA cycle.

While glycerol gets converted into glycerol phosphate by GLYCEROL KINASE and then into dihydroxyacetone phosphate (DHAP) by GLYCEROL PHOSPHATE DEHYDROGENASE.

Question 32

When stocks of carbohydrates and fatty acids are low, glucose molecules can be generated from catabolism intermediates of ______ _______ (or from any tissues).

High rate of amino acid catabolism in the muscles is rare and occurs only in certain nutritional or pathological states:

Answer 32

muscle proteins

1) Hyperproteic diets
2) Fasting
3) Type 1 diabetes (doesn’t get the insulin signal, thinks there is no glucose)

Question 33

What is the Pasteur effect?

Answer 33

When anaerobic yeast cultures are exposed to oxygen, the rate of glucose utilization DECREASES greatly.

Anaerobic glycolysis of one molecule of glucose yields 2 ATP and 2 NADH whereas the complete oxidation of 1 molecule of glucose through aerobic glycolysis will generate roughly 30-32 molecules of ATP.

Therefore, the yeast does not use as many glucose to produce energy.

Question 34

Intracellular levels of glycolytic intermediates after oxygenation are pointing towards the conversion of _____ to ______ as an important control point (PFK).

Answer 34

D-fructose-6-phosphate (F6P) to D-fructose-1,6-bisphophate (F1-6BP).

This is reaction 3 of glycolysis.

After oxygenation, there is significant less FBP and following intermediates of glycolysis. However, G6P and F6P are still high! Therefore, there must be a major regulation that occurs at reaction 3!

*this was the first regulation observed.

Question 35

(T/F) Glycolysis is controlled at the level of the three endergonic reactions of the pathway (1, 3, and 10 which are rate limiting). The regulation of gluconeogenesis will take place at the same three levels!

Answer 35

False!

Glycolysis is controlled at the level of the three EXERGONIC reactions of the pathway (1, 3, and 10 which are rate limiting). The regulation of gluconeogenesis will take place at the same three levels!

Question 36

Regulation is achieved mostly by allosteric modulators. Briefly describe allostery.

Answer 36

The change in a protein’s conformation brought about by the binding of a regulatory ligand (a site different than the catalytic site). The change in conformation alters the activity of the protein.

It can be positive or negative!
Positive: increases protein’s efficiency.
Negative: decreases protein’s efficiency.

Question 37

How is reaction 1 of glycolysis regulated?

Answer 37

Allosteric control of HEXOKINASE!

Question 38

1) How is Hexokinase I (muscle) regulated?

2) Is Hexokinase IV (glucokinase) regulated the same way?

Answer 38

1) Hexokinase I is INHIBITED by G6P (product of reaction 1).

2) No, Hexokinase IV (liver + pancreas) is not inhibited by G6P. It produces G6P at a concentration which hexokinase I would have been inhibited. This G6P can be used in other pathways; such as glycogen metabolism and Pentose Phosphate Pathway.

Question 39

What is GLUT2? What does it do?

Answer 39

The Glucose Transporter, GLUT2, establishes an equilibrium between the concentration of GLUCOSE in the blood and the hepatocytes.

If there is a high [glucose] in the blood, it gets transported into the hepatocytes.

Question 40

How is Hexokinase IV (liver + pancreas) regulated?

Answer 40

Hexokinase IV is regulated by the Glucokinase Regulatory Protein (GKRP), which sequesters hexokinase IV in the nucleus.

When the concentration of glucose increases in the (liver) cells, the increasing concentration in glucose will act as an INHIBITOR of glucokinase regulatory protein (GKRP).

Therefore, under high glucose conditions Hexokinase IV is released in the cytosol to break down glucose.

When the concentration of blood glucose drops below 5mM, fructose-6-phosphate (F6P) acts as an ALLOSTERIC ACTIVATOR of GKRP, increasing its binding to Hexokinase IV. So at low glucose conditions, Hexokinase IV is sequestered in the nucleus!

Question 41

How is reaction 11 of gluconeogenesis (corresponding to reaction 1 of glycolysis) regulated?

Answer 41

High concentration of G6P positively regulates glucose-6-phosphatase into the conversion of glucose-6-phosphate (G6P) into glucose as a SUBSTRATE LEVEL CONTROL.

This is because the glucose-6-phosphatase Km (2-3mM) is higher than the intracellular concentration of G6P (0.05-0.1mM); activity of enzyme increases in relation to the increase in G6P concentration.

Question 42

Phosphofructokinase (PFK), the enzyme of reaction 3 of glycolysis, is inhibited by _____ ______ ______.

Answer 42

Energy Level Sensor

*is there lots of ATP? Acetyl-CoA?
*example: if a muscle is working a lot and thus using ATP, AMP and ADP will be made. This causes the enzyme to produce more ATP. However, if a muscle isn’t using as much ATP and there is a surplus, the enzyme slows down.

Question 43

(T/F) Phosphofructokinase (PFK1) is inhibited by ATP and citrate but activated by AMP, ADP and fructose 2,6-bisphosphate.

Answer 43

True!

*binding of AMP/ADP and fructose 2,6-bisphosphate changes the catalytic pocket of PFK to make it a better fit for the substrate.

Question 44

ATP is a _______ and an ______; it binds to the active site as well as an allosteric site on PFK.

In the presence of an excess of ATP, ATP binds to an ________ site and PFK activity is _________.

When the energy charge (ATP) is low, PFK will be _______ by the binding ______ to the allosteric site.

High ATP: _____ Glycolysis
Low ATP: _____ Glycolysis

Answer 44

Substrate; inhibitor

Allosteric; Inhibited

Activated; ADP

Low; High

Question 45

(T/F) Citrate is a good indicator of the level of energy charge in the cell. An elevated [ ] of citrate, which is an intermediate of the TCA cycle, is an indication of a low energy charge and therefore, it will inhibit PFK’s activity.

Answer 45

False!

Citrate is a good indicator of the level of energy charge in the cell. An elevated [ ] of citrate, which is an intermediate of the TCA cycle, is an indication of a HIGH energy charge and therefore, it will inhibit PFK’s activity.

Question 46

Briefly answer the questions regarding F2,6BP:

1) What is Fructose 2,6-bisphosphate (F2,6 BP)?

2) Which enzyme is it generated by?

3) What is its role in glycolysis?

4) How is it different than other allosteric substrates of PFK1?

Answer 46

1) Fructose 2,6 bisphopshate (F2,6BP) is a positive allosteric modulator of PFK1.

2) F2,6BP is generated by the PFK/FBPase-2 enzyme. (Bifunctional PFK-2/FBPase-2 is distinct from than the ones used for glycolysis and gluconeogenesis)

3) PFK1, the enzyme used in reaction 3 of glycolysis, is positively regulated by this molecule. Absence of it results in a decrease in activity of PFK1; less glycolysis occurring.

4) While other allosteric substates of PFK1 such as ATP, AMP are within the cell, this molecule is controlled by hormones, thus includes the whole body.

Question 47

Reaction 3 of glycolysis corresponds to reaction ____ of gluconeogenesis.

While PFK1 (enzyme in reaction 3 of glycolysis) is positively regulated by fructose-2, 6-bisphosphate, fructose-1,6-biphosphatase (enzyme in reaction 9 of gluconeogenesis) is ________ regulated.

Answer 47

9

Negatively

Question 48

Increase in [F26BP] ________ glycolysis and _______ gluconeogenesis.

Decrease in [F26BP] ________ glycolysis and _______ gluconeogenesis.

Answer 48

Stimulates; inhibits

Inhibits; Stimulates

*glycolysis and gluconeogenesis is controlled by this hormone in a reciprocal fashion

Question 49

1) What enzyme does insulin (high glucose) positively regulate?

2) What enzyme does glycogen (low glucose) positively regulate?

Answer 49

1) Phospho-protein phosphatase (PPP)

2) cAMP-dependent protein kinase (PKA)

Question 50

The FBPase-2 activity of PFK/FBPase-2 _________ Fructose 2,6-bisphosphaye.

The PFK-2 activity of PFK/FBPase-2 _________ Fructose 2,6-bisphosphaye.

Answer 50

Degrades to Fructose 6-phosphate

Synthesizes from Fructose 6-phosphate

Question 51

1) What happens to the concentration of F26BP when insulin is present?

2) What happens to the concentration of F26BP when glucagon is present?

Answer 51

1) Insulin increases the activity of Phospho-protein phsophatase (PPP). This enzyme removes the phosphate on PFK/FBPase-2, causing the PFK-2 to be active while FBPase-2 is inactive. This INCREASES the concentration of F26BP.

2) Glucagon (high cAMP) increases the activity of cAMP-dependent protein kinase (PKA), which phosphorylates PFK/FBPase-2, causing the FBPase-2 subunit to be active and PFK-2 to be inactive. This DECREASES the concentration of F26BP.

*high glucose; want to stimulate glycolysis
*low glucose; want to stimulate gluconeogenesis

Question 52

How is reaction 10 of glycolysis regulated?

Answer 52

Allosteric control of the pyruvate kinase.

Pyruvate kinase is inhibited by Acetyl-CoA and by ATP (energy sensor).

It is activated by fructose-1,6-biphosphate (F1,6BP).

Question 53

The liver pyruvate kinase is _________ by cAMP-dependent phosphorylation (upon glucagon release).

Answer 53

inactivated

*pyruvate kinase is regulated by glucagon

Question 54

How is reaction 1 of gluconeogenesis regulated?

Answer 54

Pyruvate carboxylase (enzyme that turns pyruvate into oxaloacetate) is activated by Acetyl-CoA.

Acetyl-CoA acts as a master regulator of pyruvate’s fate.

Question 55

What is a metabolic futile cycle?

What is its purpose?

Answer 55

Metabolic futile cycle is one in which a precursor is converted into a product by a forward reaction and then resynsthesized to the precursor. In such a reaction, there is no net product accumulation, but energy (ATP) is used.

An example of it would be if glycolysis and gluconeogenesis occur at the same time.

Homeostatic purpose: metabolic idling + a treatment for obesity (burn glucose and spend ATP at the same time doing it)?