Title: BV2 / BV2 - 2 / Modul 5 / Gluconeogenesis

Q: How can liver cells and kidney cells take care of pyruvate and lactate?
A: Hepatocytes and kidney cells can also transform pyruvate and lactate back into glucose via a process called gluconeogenesis from non-carbohydrate sources, as well as from other non-carbohydrate sources, such as glycerol or alanine

Q: Does glycolysis and gluconeogenesis take place at the same time?
A: Gluconeogenesis and glycolysis usually do not take place at the same time and are in a tightly regulated balance

Q: What is gluconeogenesis?
A: Gluconeogenesis is the synthesis of glucose molecules from non-carbohydrate precursors that can be converted to pyruvate or intermediates of glycolysis, such as lactate, glycerol, alanine, and glutamine.

Q: Which substrates can be used in gluconeogenesis?
A: - Lactate
- Glycerol
- Alanine
- Glutamine

Q: Where in the body does gluconeogenesis occur?
A: This process predominantly occurs in the liver but to some extent also in the renal cortex.

Q: What does gluconeogenesis do together with glycogen breakdown?
A: Together with glycogen breakdown gluconeogenesis maintains blood glucose levels during periods of fasting, starvation, or exercise.

Q: What else does gluconeogenesis provide the body with?
A: Gluconeogenesis also provides the body with a means to regenerate glucose from lactate, released during anaerobic glycolysis

Q: What is the process called when gluconeogenesis in the liver regenerates glucose from lactate formed under anaerobic glycolysis?
A: The Cori cycle

Q: What is the Cori cycle important for?
A: Important for regulating blood glucose levels and blood
pH.

Q: What is the Cori cycle?
A: When lactate from anaerobic glycolysis goes to the liver, via the bloodstream, where gluconeogenesis converts it back to glucose and sends it back to the cells via the blood, and then the process can be repeated

Q: How much does gluconeogenesis share with glycolysis?
A: Gluconeogenesis shares seven out of the 10 enzymatic reactions from glycolysis, except for steps 1, 3 and 10 of glycolysis

Q: Which steps does gluconeogenesis not share with glycolysis?
A: Steps 1,3, and 10 of glycolysis

Q: Why are steps 1,3, and 10 of glycolysis not reversed/ why are they different in gluconeogenesis?
A: These reactions are highly exergonic during glycolysis and are therefore replaced by more energy favorable reactions during gluconeogenesis.

Q: How does the step 10 reaction happen i gluconeogenesis?
A: Pyruvate is phosphorylated to phosphoenolpyruvate via an oxaloacetate intermediate step, catalysed by the 2 enzymes pyruvate carboxylase and PEP carboxykinase

Q: How does the step 3 reaction happen i gluconeogenesis?
A: Fructose-1,6-bisphosphate is dephosphorylated by fructose-1,6-bisphosphatase

Q: At which step does gluconeogenesis terminate in other cells than the liver?
A: At the formation of glucose-6-phosphate

Q: At which step does gluconeogenesis terminate in liver cells?
A: Glucose

Q: Why does gluconeogenesis terminate at the formation of glucose in the liver, but at the formation of glucose-6-phosphate in other cells?
A: Because the liver needs to be able to release it back into the blood stream for other cells to use, if glucose is phsophorylated it doesn't work

Q: Where in the liver cells is glucose-6-phosphate converted inot glucose?
A: Glucose 6-phosphate is further converted to glucose inside the lumen of the endoplasmic reticulum by glucose 6-phosphatase

Q: Which enzyme catalyses the formation of glucose from glucose-6-phosphate in liver cells?
A: Glucose-6-phosphatase

Q: What is the overall reaction of gluconeogenesis?
A: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP + 4H 2 O + 2H + → glucose + 2 NAD + + 4 ADP + 2 GDP + 6P i

Q: What regulates gluconeogenesis and glycolysis rescpectively in a high energetic state?
A: In a high energetic state high levels of acetyl-CoA and citrate stimulate gluconeogenesis, while high ATP, citrate, glucose 6-phosphate and alanine levels inhibit glycolysis
- Gluconeogenesis activated (Acetyl-CoA, Citrate)
- Glycolysis inhibited (ATP, Citrate, Glucose-6-phosphate, alanine)

Q: What regulates gluconeogenesis and glycolysis rescpectively in a low energetic state?
A: During low energy levels, the higher levels of AMP, fructose  2,6-bisphosphate and fructose 1,6-bisphosphate stimulate glycolysis, while AMP, ADP, and fructose 2,6-bisphosphate inhibit gluconeogenesis.
- Glycolysis activated (AMP, fructose-2,6-bisphosphate, fructose-1,6-bisphosphate)
- Gluconeogenesis inhibited (AMP, ADP, fructose-2,6-bisphosphate)

Q: Why is the balance of glycolysis and gluconeogenesis important in liver cells?
A: Liver cells are responsible for the regulation and maintenance of blood glucose levels and therefore need to balance gluconeogenesis and glycolysis

Q: What is a central enzyme to regulate glycolysis and gluconeogenesis in the liver?
A: A central enzyme in this regulation is the bifunctional allosteric enzyme phosphofructokinase 2 ( PFK2 )

Q: What does phosphofructokinase-2 (PFK-2) consist of?
A: This enzyme has both a kinase (PFK2) as well as phosphatase (fructose bisphosphatase 2 ; FbP2) domain, which are in opposing states (active / inactive) upon phosphorylation

Q: What happens with the different domains of PFK-2 upon phosphorylation?
A: They are in opposing states (active/inactive), and which one will e active depends on phosphorylation

Q: What are the fucntions of the 2 domains PFK2 and FbP2 in the PFK-2 enzyme?
A: The function of PFK2 and FbP2 is to phosphorylate or dephosphorylate fructose 6-phosphate to fructose 2,6-bisphosphate or visa versa

Q: What does PFK2 do?
A: Phosphorylates fructose-6-phosphate into fructose-2,6-bisphosphate

Q: What does FbP2 do?
A: Dephosphorylated fructose-2,6-bisphosphate into fructose-6-phosphate

Q: What is fructose-2,6-bisphosphate?
A: Fructose 2,6-bisphosphate is a potent allosteric activator of glycolysis and inhibitor of gluconeogenesis

Q: What happens of PFK2 is active?
A: It phosphorylates fructose-6-phosphate into fructose-2,6-bisphosphate, which in turn allosterically activates glycolysis and inhibits glukoneogenesis

Q: What happens of FbP2 is active?
A: It dephosphorylates fructose-2,6-bisphosphate into fructose-6-phosphate, which in turn activates gluconeogenesis but inhibits glycolysis

Q: What happens with PFK2 under high blood glucose levels?
A: Under high blood glucose levels insulin stimulates the enzyme  phosphoprotein phosphatase  to dephosphorylate PFK2 activating its kinase activity.

Q: Which protein dephosphorylates PFK2?
A: Phosphoprotein phosphatase

Q: What does dephosphorylation do to PFK2?
A: Activates its kinase activity - PFK-2

Q: What does phosphorylation do to PFK2?
A: Activates FbP2

Q: What does high blood glucose levels lead to?
A: High blood glucose leads to increased fructose 2,6-bisphosphate levels and the activation of glycolysis by stimulating phosphofructokinase  activity

Q: What happens during low blood glucose levels (glucagon)?
A: Under low blood glucose levels glucagon activates a signal transduction cascade that leads to the formation of cAMP

Q: What does glucagon activated cAMP do?
A: cAMP activates the enzyme Protein kinase A , which phosphorylates PFK2 and thereby inactivates PFK2and activating FbP2

Q: Which enzyme phosphorylates PFK2?
A: Protein kinase A (PKA)

Q: What does activated FbP2 do?
A: Activated FbP2 leads to the dephosphorylation of fructose 2,6-bisphosphate back to fructose 6-phosphate.
- Inhibition of glycolysis, activation of gluconeogenesis

Q: What is the process of gluconeogenesis?
A: Gluconeogenesis is the process by which the cells of our body can actually generate ATP molecules starting from precursor non-carbohydrate molecules, such as pyruvate, lactate, amino acids and glycerol

Q: Which cells typically carry out the process of gluconeogenesis?
A: - The cells in our body that actually are responsible for regulating and maintaining glucose levels in our blood, such as
  - Liver cells
  - Kidney cells - to a smaller extent
- These cells carry out gluconeogenesis

Q: Are liver and kidney cells the only cells that can preform gluconeogenesis?
A: Under certain conditions cells such as skeletal muscle cells, cardiac muscle cells, and brain cells can also carry out gluconeogenesis

Q: What can be said about gluconeogenesis in different cells?
A: So this process isn't exclusive to liver and kidney cells, other cells also use this process
- But there is an important difference

Q: WHere does step 1 of gluconeogenesis take place?
A: Takes place within the matrix of the mitochondria

Q: Why does step 1 of gluconeogenesis take place in the matrix of the mitochondria
A: Because the enzyme that carries out step 1 is actually found within the matrix of  mitochondria

Q: WHat happens to the pyrvate in the matrix of the mitochondria in gluconeogenesis?
A: In the matrix, a pyruvate molecule is transformed into an oxaloacetate intermediate and it's catalysed by a pyruvate carboxylase

Q: WHat does pyruvate carboxylase do?
A: This enzyme hydrolyses an ATP molecule and the energy that is released in that hydrolysis is used to drive the carboxylation of pyruvate into and oxaloacetate
Hence, we break down an ATP to attach a CO2 to pyruvate to form oxaloacetate

Q: WHat do we break down to form oxaloacetate from pyruvate?
A: We break down an ATP to attach a CO2 to pyruvate to form oxaloacetate
Because we take 2 pyruvate to transform them into 2 oxaloacetate we use 2 ATP and 2 CO2 molecules in step 1

Q: WHat happens in gluconeogenesis as soon as we have formed oxaloacetate in step 1?
A: Once we have formed the oxaloacetate we can move on to step 2

Q: WHat is the problem with step 1- step 2 in gluconeogenesis?
A: The problem with step 1- step 2 is that step 2 takes place in the cytoplasm, because the enzyme used to catalyse step 2 is found in the cytoplasm

Q: What do we have to do with oxaloacetate before it can enter step 2 in the cytoplasm in gluconeogenesis?
A: What that means is that before oxaloacetate can be used in the reaction's step 2 it must be moved into the cytoplasm

Q: Can oxaloacetate be moved to the cytoplasm from the mitochondria?
A: The oxaloacetate itself cannot be moved across the 2 membranes of the mitochondria

Q: What has to happen to the oxaloacetate before it can enter the cytoplasm?
A: Before it can be moved it has to be reduced

Q: How is oxaloacetate reduced?
A: So, we reduce oxaloacetate into malate through the enzyme malate dehydrogenase and it uses an NADH molecule (2 in total for the combined of the 2 oxaloacetates)

Q: What happens to the malate formed from oxaloacetate in gluconeogenesis?
A: The malate then moves across special membrane proteins found in both membranes in the mitochondria and into the cytoplasm

Q: Which enzyme transforms oxaloacetate into malate?
A: Malate dehydrogenase

Q: WHat happens to the malate in the cytoplasm in gluconeogenesis?
A: In the cytoplasm we use that same malate dehydrogenase but now we use the NAD+ to oxidise the malate back into the oxaloacetate

Q: WHat can be said about the use of NADH/NAD+ in the conversion between oxaloacetate and malate?
A: We essentially use 2 NADHs to form 2 NAD+s and then out in the cytosol we use 2 NAD+ to form 2 NADHs

Q: How many oxaloacetate molecules do we form in the cytoplasm in gluconeogenesis?
A: We form 2 oxaloacetate molecules in the cytoplasm, essentially the same ones we had inside the mitochondria (matrix)

Q: Which enzyme is used in step 2 of gluconeogenesis?
A: In step 2 we have an enzyme known as phosphoenolpyruvate carboxykinase (PEP carboxykinase)

Q: What does PEP carboxykinase do in step 2 of gluconeogenesis?
A: What this enzyme basically does is that it phosphorylates the oxaloacetate, but at the same time it decarboxylates the oxaloacetate to form the phosphoenolpyruvate

Q: Why do we need these 2 different reactions in the same step (step 2 of gluconeogenesis)?
A: Because to phosphorylate that molecule that requires energy and when we decarboxylate something we essentially remove 1 carbon dioxide group that releases energy - So we couple these 2 reactions

Q: How do we couple the 2 reactions in step 2 of gluconeogenesis?
A: We use the decarboxylation reaction to drive the phosphorylation and we produce the 2 phosphoenolpyruvate from the 2 oxaloacetate

Q: What is the difference in use of energy molecules between step 1 and step 2 in gluconeogenesis?
A: In step 1 we used ATP molecules form energy but in step 2 we used GTP molecules

Q: How many GTP are used in step 2 of gluconeogenesis?
A: So, a single GTP molecule is used per oxaloacetate that is used to produce the phosphoenolpyruvate

Q: How can step 1&2 od gluconeogenesis be summarized?
A: In step 1 and 2 we transformed the pyruvate into phosphoenolpyruvate

Q: HOw is step 1&2 of gluconeogenesis similar to step 10 in glycolysis?
A: We know that in step 10 of glycolysis we form pyruvate from the phosphoenolpyruvate

Q: If we have the same products and substartes but reversed in step 1&2 of gluconeogenesis as in step 10 of glycolysis, why
can't we use the reversed process of forming that phosphoenolpyruvate from the
pyruvate the same way as in glycolysis?
A: Because the reversed step 10 of glycolysis would be a very endergonic process
So, we see to bypass that endergonic process, we create a completely different type of reaction pathway that actually creates a process that is favourable

Q: How do be basically avoid the reversed endergonic step 10 of glycolysis in step 1&2 of gluconeogenesis?
A: So, we basically drive the unfavourable process by using these favourable processes such as the hydrolysis of ATP in step 1 and the decarboxylation in step 2

Q: What can be said about steps 3-7 in gluconeogenesis?
A: All these steps are essentially the same exact steps as the ones in glycolysis, except that they are in reverse

Q: WHy is it that we can use steps 3-7 from glycolysis but reversed in gluconeogenesis?
A: Because these steps have a Gibbs free energy value that is very close to 0 - what that means is that these are at equilibrium
So, if we are at the right conditions inside the cell, if they favour the formation of glucose/gluconeogenesis, these steps will simply take place spontaneously
That's why gluconeogenesis utilises the same steps as glycolysis, except they are in reverse because these steps are at equilibrium

Q: Do we use the same enzymes for steps 3-7 in gluconeogenesis as in glycolysis?
A: Yes, we
use the same enzymes in gluconeogenesis as in glycolysis for step 3-7

Q: What happens in step 3 of gluconeogenesis?
A: Phosphoenolpyruvate is transformed into 2-phosphoglycerate by enolase

Q: What happens in step 4 of gluconeogenesis?
A: Then the phosphoglycerate mutase transfers the phosphoryl group from carbon 2 to carbon 3 to produce 3-phosphoglycerate

Q: What happens in step 5 of gluconeogenesis?
A: The phosphoglycerate kinase creates 1,3-BPG from previous step

Q: What happens in step 6 of gluconeogenesis?
A: - Via the action of GAP dehydrogenase 1,3-BPG is transformed into the 2 glyceraldehydes
- One of those 2 glyceraldehydes is transformed into the dihydroxyacetone phosphate (DHAP) by the action of triosephosphate isomerase

Q: What happens in step 7 of gluconeogenesis?
A: Now that we have formed GAP and DHAP aldolase can combine these 2 molecules in step 7 to form fructose-1,6-bisphosphate

Q: What happens once we get to step 8 of gluconeogenesis?
A: Once
we come to step 8 the pathway becomes different from the pathway we use in
glycolysis

Q: Why is step 8 of gluconeogenesis not the reverse of the step in glycolysis?
A: Because in glycolysis when we go from fructose-6-phosphate to fructose-1,6-bisphosphate that process is very exergonic
That means that if we would simply use the reverse process of glycolysis that would mean that the reversed reaction would be a very endergonic reaction

Q: Which enzyme do we use in step 8 of gluconeogenesis?
A: We use fructose-1,6-bisphosphatase (phosphatase meaning we remove a phosphoryl group)

Q: What happens in the reaction in step 8 of gluconeogenesis?
A: What happens in this reaction is that we use an H2O molecule to hydrolyse the ester bond that is holding carbon-1 and the phosphoryl group

Q: Thermodynamically, what type of process takes place in step 8 of gluconeogenesis?
A: This
process actually releases energy - it's an exergonic process

Q: What does it entail that the step 8 reaction in gluconeogenesis is exergonic?
A: That we are able to form the fructose-6-phosphate spontaneously under physiological cell conditions

Q: What is step 9 of gluconeogenesis?
A: Once we form that fructose-6-phosphate, step 9 is basically the reverse of step 2 in glycolysis

Q: Why is step 9 in gluconeogenesis basically the reverse of step 2 in glycolysis?
A: Again, the same reason as for steps 3 through 7
Because this step is reversible and Gibbs free energy is very close to 0, meaning this reaction is at equilibrium and will spontaneously take place and will form glucose-6-phosphate

Q: What happens in step 9 of gluconeogenesis?
A: Phosphoglucose isomerase isomerases fructose-6-phosphate into glucose-6-phosphate

Q: What happens in step 10 of gluconeogenesis in liver och kidney cells?
A: Since the role of these cells is to regulate the glucose levels inside our blood, their job is to form glucose and release these into the blood when our blood glucose levels drop
- So, what the next step would be in a liver or kidney cell is that the glucose-6-phosphate is moved into the lumen of the endoplasmic reticulum
- Once that G-6-P is inside the lumen, we have the hydrolysis of the ester bond between the phosphoryl group and the carbon by the activity of glucose-6-phosphatase
  - A protein known as calcium binding stabilising protein assists the glucose-6-phosphate in this hydrolysis process
  - The H2O is found in the lumen of the ER and we hydrolyse the ester bond to form an inorganic phosphate group (Pi) as well as the glucose

Q: How does th glucose-6-phosphate move into the ER in step 10 of gluconeogenesis?
A: In liver or kidney cells the G-6-P moves into the lumen of the ER through via a special type of membrane protein called T1 - glucose-6-phosphate transporter

Q: How does the Pi move into the cytosol after step 10 of gluconeogenesis?
A: We have a special protein in the membrane of the ER known as T2 - phosphate transporter, which basically moves that inorganic phosphate Pi into the cytoplasm

Q: How does the glucose from gluconeogenesis exit the ER?
A: We also have a special glucose transporter called T3 in the membrane of the ER, that shuttles the glucose into the cytoplasm

Q: Which transporter transports the glucose-6-phosphate into the ER in gluconeogenesis?
A: T1 - glucose-6-phosphate transporter

Q: Which transporter transports the Pi into the cytosol from the ER after gluconeogenesis?
A: T2 - phosphate transporter

Q: Which transporter transports the glucose into the cytosol from the ER after gluconeogenesis?
A: T3

Q: When does gluconeogenesis differ between liver/kidney cells and other cells?
A: After step 9 is where the difference between the different types of cells comes into play
- Basically, step 10 is different

Q: What's the reaction in step 10 in gluconeogenesis in liver/kidney cells?
A: In the ER, hydrolysis of the ester bond between the phosphoryl group and the carbon by the activity of glucose-6-phosphatase makes glucose-6-phosphate into glucose
- A protein known as calcium binding stabilising protein assists the glucose-6-phosphate in this hydrolysis process

Q: WHy does step 10 of gluconeogenesis in liver cells occur in the ER?
A: The H2O is found in the lumen of the ER and we hydrolyse the ester bond to form an inorganic phosphate group (Pi) as well as the glucose

Q: Which helper protein assists the glucose-6-phosphatase ins step 10 of gluconeogenesis in liver cells?
A: A protein known as calcium binding stabilising protein assists the glucose-6-phosphatase in this hydrolysis process

Q: What else is involved in transforming G-6-P into glucose in step 10 of gluconeogenesis?
A: We see that five different proteins are involved in transforming the G-6-P into glucose in liver and kidney cells

Q: Which proteins are involved in transforming G-6-P into glucose in step 10 of gluconeogenesis?
A: - T1 - glucose-6-phosphate transporter
- Glucose-6-phosphatase
- Ca2+ binding stabilising protein
- T2 - phosphate transport
- T3 - glucose transport

Q: What happens when we have formed glucose from gluconeogenesis in liver cells?
A: Now that we have formed the glucose and released it into the cytoplasm it can easily leave the cell via a special type of membrane glucose transporter

Q: What's the difference between glucose-6-phosphate and glucose?
A: G-6-P, because of that phosphate group, cannot actually leave the cell while glucose can

Q: Why does gluconeogenesis occur in liver cells?
A: The reason this process takes place in liver cells is so that we can remove that phosphoryl group that traps the glucose inside the cells

Q: What does step 10 of gluconeogenesis look like in muscle cells?
A: In
cells like muscle cells, that are not responsible for maintaining the glucose
levels in our blood, this process will stop at the glucose-6-phosphate stage

Q: What happens once G-6-P is formed in gluconeogenesis in liver cells?
A: Once the muscle generates G-6-P it doesn't want to remove that phosphoryl group because it doesn't want to lose that glucose to the outside

Q: Why don't we wanna form glucose from gluconeogenesis in muscle cells?
A: Because, once we have formed glucose that glucose is no longer trapped inside the cell, and we want it in the muscle cell

Q: What can happen with the formed G-6-P form gluconeogenesis in muscle cells?
A: So, in cells like skeletal muscle cells or cardiac muscle cells once we form the G-6-P this process stops - The G-6-P can in turn be used to form glycogen or be used to form ATP molecules

Q: Which other molecules can enter gluconeogenesis apart from pyruvate?
A: - Amino acids
- Glycerol
- Lactate

Q: How does amino acids and lactate enter gluconeogenesis?
A: Lactate and certain amino acids such as alanine can actually be transformed into pyruvate

Q: Via what does aminoacids and lactate enter gluconeogenesis?
A: These
enter the gluconeogenic pathway/gluconeogenesis via pyruvate

Q: What can other aminoacids that are used in gluconeogenesis be transformed into apart for pyruvate?
A: Other
amino acids can be transformed into oxaloacetate

Q: How do glycerol enter gluconeogenesis?
A: Glycerol molecules are transformed into DHAP - dihydroxyacetone phosphate molecules and they enter the pathway at the corresponding step

Q: What is the net reaction formula for gluconeogenesis?
A: 2 pyruvate + 4ATP + 2 GTP + 2NADH + 6H2O --> glucose + 4ADP + 2GDP + 6 Pi + 2 NAD+ + 2H+

Q: What type of reaction is gluconeogenesis under normal physiological conditions?
A: This reaction under normal physiological condition is an exergonic reaction - It releases a certain amount of energy

Q: What do we basically do in gluconeogenesis?
A: Basically, in this process we use the hydrolysis of these high energy molecules (ATP and GTP) to drive all the other unfavourable processes, so at the end this reaction takes place spontaneously, quickly and effectively inside the cells of our bodies

Q: How are gluconeogenesis and glycolysis regulated together?
A: These 2 processes are regulated in a reciprocal fashion, meaning when one of these processes occurs in our bodies the other is turned off

Q: Why is it that gluconeogenesis and glycolysis can't take place in the cell at the same time?
A: Actually they can take place at the same time if we examine energy values
- So, there is actually no energy barrier which is inhibiting these processes from happening at the same time

Q: Why is there no energy barrier keeping glycolysis and gluconeogenesis from happening at the same time?
A: Because both processes release a certain amount of energy into the cell ->That means that they are both spontaneous exergonic reactions

Q: What would happen if we would sum up the glycolysis and gluconeogenesis reactions?
A: If we would sum up the reactions they would create a net exergonic reaction, since both are exergonic

Q: What is it that actually prevents glycolysis and gluconeogenesis from happening at the same time?
A: What actually prevents these reactions for happening at the same time are the special allosteric enzymes

Q: What does the cell use to prevent glycolysis and gluconeogenesis from happening at the same time?
A: The cell uses special allosteric enzymatic regulation to prevent these processes to take place at the same time

Q: Why does the cell wanna prevent the processes from happening at the same time?
A: - Gluconeogenesis, although we release a certain amount of energy, it's an exergonic process,  we use up 4 ATP and 2 GTP
- While in glycolysis, we produce a net amount of 2 ATP molecules
So, if we sum up these reactions we can see that the cell uses up 2 ATP molecules and 2 GTP, meaning that when glycolysis takes place at the same time as gluconeogenesis we are not going to form any ATP molecules, we are going to use them up, hence, defeating the purpose of glycolysis -> that's why these processes don't take place at the same time in our cells

Q: How is this actually achieved - how can the cell regulate glycolysis and gluconeogenesis?
A: - It's the fact that our cells have these allosteric enzymes that can be used to regulate this process
- There are 2 points along this pathway, 1 & 2, where we have allosteric enzymes that can regulate the pathways of gluconeogenesis and glycolysis

Q: What does gluconeogenesis and glycolysis use to make energy/glucose, respectively?
A: Gluconeogenesis uses pyruvate molecules to form energy/glucose, while glycolysis uses glucose to form pyruvate

Q: What happens with glycolysis and gluconeogenesis at a high cellular energy charge?
A: - In this case, glycolysis will be shut off as we don't need to make more ATP
- When there is an excess of ATP can be used by gluconeogenesis (uses 4 ATP and 2 GTP) to produce glucose which we then can use to form glycogen, form glycogen storages in our cells

Q: What regulates the process of gluconeogenes turning on at a high energy charge?
A: Basically pyruvate carboxylase and fructose-1,6-bisphoaphatase are activated and that allows the process of gluconeogenesis to occur

Q: Why is Acetyl-CoA an activator for pyruvate carboxylase?
A: Acetyl-CoA is needed to allow the binding between the CO2 and the biotin component of the pyruvate carboxylase

Q: What activates fructose-1,6-bisphosphatase in gluconeogenesis?
A: In the step of converting fructose-1,6-bisphosphate into fructose-6-phosphate, citrate activates fructose-1,6-bisphosphatase

Q: Why does citrate activate fructose-1,6-bisphosphatase in gluconeogenesis?
A: - The citrate is an agent tells the cell that we have plenty of molecules to go around in the TCA - citrate being an intermediate in the cycle -> so we don't want to produce more intermediates in the TCA, hence we don't want to produce more pyruvate
  - This implies that pyruvates now can be used in gluconeogenesis to form glucose, which in turn can be used to replenish our glycogen storages

Q: Why does acetyl-CoA activate fructose-1,6-bisphosphatase in gluconeogenesis?
A: - Acetyl-CoA is also needed for the TCA cycle to take place, so if we have many Acetyl-CoA it means we have many molecules needed for the citric acid cycle but the excess indicates that the TCA doesn't need any more Acetyl-CoA - pyruvates are used to produce acetyl-CoA
  - Hence, high levels of acetyl-CoA means we don't want to produce more pyruvate molecules, so we can use the pyruvate to form glucose via gluconeogenesis -> its rate increases

Q: What happens with glycolysis at a high energy charge?
A: When we have a high energy charge in the cell glycolysis will be turned off

Q: How will glycolysis be turned off at a high energy charge?
A: - Phosphofructokinase is inhibited by ATP (when there is a high conc of it) and the ATP will bind onto the allosteric site of phosphofructokinase inhibiting its activity
- At the same time as this is inhibited the fructose-6-phosphate cannot be transformed into fructose-1,6-bisphosphate, leading to an increasing accumulation of fructose-6-phosphate in the cell
- As this conc builds up and since F-6-P and G-6-P are in equilibrium the conc of G-6-P in the cell will increase as well
- As the G-6-P builds up the G-6-P goes on to inactivate the hexokinase
  - So, by inactivating phosphofructokinase we also inactivate hexokinase

Q: What happens in mucsle cells when the rate of glycolysis exceeds the rate of oxidative phosphorylation?
A: In skeletal muscle, when the
rate of glycolysis exceeds the rate of oxidative phosphorylation there
will be a build-up of lactic acid, as a result of lactic acid fermentation

Q: What will lactic acid fermentation in skeletal muscle cause?
A: This will lead to an increase in H+ ions and that will also inhibit the function of phosphofructokinase

Q: What affects the function of PFK in liver cells instead of H+?
A: Citrate affects the function of PFK

Q: What does citrate do to inhibit PFK in liver cells?
A: In liver cells citrate goes on and binds to PFK and "tells" it to arrest glycolysis

Q: Why does citrate inhibit PFK in liver cells?
A: Because if we have plenty of ATP molecules it also means we have plenty of pyruvate molecules and pyruvate goes on to the TCA and there forms citrate
- So if we have lots of citrate molecules we don't want more pyruvate so the citrate goes on to PFK and binds to it inhibiting it

Q: What does pyruvate kinase catalyse?
A: Catalyses the last step in glycolysis

Q: What inhibits pyruvate kinase?
A: - ATP inhibits pyruvate kinase
- In addition, since pyruvate is used to form alanine
  - If we have plenty of ATP -> plenty of pyruvate -> plenty of alanine

Q: What will alanine do at high concentrations?
A: The alanine will at high concentrations then go back and create a negative feedback loop and inhibit the action of pyruvate kinase

Q: What else affects the pyruvate kinase found in liver cells?
A: In liver cells the pyruvate kinase exists in the L-isozyme form and the activity of this form is also affected by phosphorylation

Q: What happens with glycolysis and gluconeogenesis at high ATP concentrations?
A: At a high ATP conc in cells we don't want to form more ATP, so glycolysis is turned off as gluconeogenesis is turned on

Q: What can we use in gluconeogenesis?
A: In gluconeogenesis we can use, pyruvate, lactate, glycerol and amino acids to form glucose

Q: What can we do with the glucose produced by gluconeogenesis?
A: We can then turn the glucose into glycogen and replenish our glycogen storages

Q: What happens with the cell at a high energy charge?
A: When the cell has an high energy charge, this tells the cell to stop producing ATP via glycolysis and begin generating glucose via gluconeogenesis to store it as glycogen

Q: What gets activated and inhibited at high ATP conc?
A: Under these conditions glycolysis is halted and gluconeogenesis is activated

Q: What is activated vs ihibited enzyme wise and by what at a high energy charge?
A: - Phosphofructokinase is inhibited by ATP, H+ (muscle), and citrate (liver)
- Hexokinase is inhibited by G-6-P
- Pyruvate kinase is inhibited by ATP, alanine, and phosphorylation (liver)
- Fructose-1,6-bisphosphatase is activated by citrate
- Pyruvate carboxylase is activated by acetyl-CoA

Q: What is activated vs ihibited enzyme wise and by what at a low energy charge?
A: - PFK is activated by AMP and Fructose-2,6-bisphosphate
- Pyruvate kinase is activated by fructose-1,6-bisphosphate
- Fructose-1,6-bisphosphatase is inhibited by ADP and fructose-2,6-bisphosphate
- PEP carboxykinase is inhibited by ADP
- Pyruvate carboxylase is inhibited by ADP

Q: What happens in the cell at a low energy charge?
A: When the cell has a low energy charge, this will tell the cell to produce more ATP via glycolysis and stop using ATP to form glucose via gluconeogenesis

Q: Which process will be inhibited and which will be activated during a low energy charge?
A: Under such conditions, glycolysis is activated and gluconeogenesis is inhibited

Q: How is it made sure that pyruvate kinase will use the frutose-1,6-bisphosphate instead of fructose-1,6-phosphatase turning it back into fructose-6-phosphate?
A: If PFK is activated we will produce more fructose-1,6-bisphosphate, and to make sure the pyruvate kinase can use them and not the fructose-1,6-phosphatase pyruvate kinase is activated by F-1,6-BP, while F-1,6-phosphatase will be inhibited by the same factors that activate PFK (AMP and F-26,-BP)

Q: What is the conclusion about when glycolysis and gluconeogenesis will be activated, respectively?
A: - When ATP is plentiful, gluconeogenesis predominates
- When ATP is scarce, glycolysis predominates

Q: Can we use Le Châtelier's principle to explain why either process takes place?
A: We cannot use this principle to explain why either process takes place

Q: Why can't we use Le Châtelier's principle to explain why either process takes place?
A: - Because the principle is used strictly for those reactions that are at equilibrium and if a reaction is at equilibrium that means Gibbs free energy in that process is 0
- But we know that in gluconeogenesis and glycolysis these processes release Gibbs free energy - are exergonic -> means that none of these processes are at equilibrium -> so we cannot use Le Châtelier's principle to explain why this process takes place and the other one doesn't

Q: What is one of the roles of our liver cells and our kidney cells?
A: One of the roles of our liver cells and our kidney cells (to a smaller extent) is to regulate and maintain proper blood glucose levels

Q: Why is the regulation of blood glucose important?
A: Because the cells of our
bodies use that glucose to form ATP molecules , which are then used to
power many different processes, for instance gluconeogenesis

Q: How can the extent of gluconeogenesis in liver/kidney cells be compared to that of other cells?
A: - Liver cells and kidney cells can undergo gluconeogenesis, to a very large extent compared to other cells
- Because other cells such as brain cells and muscle cells don't use gluconeogenesis, only to a very small extent when they need to

Q: In which celltype does gluconeogenesis not occur at all?
A: Red
blood cells cannot use gluconeogenesis at all because RBCs don't have
mitochondria

Q: What are the RBC highly dependent on due to their lack of mitochondria?
A: Due to this the RBCs highly depend on the glucose found in blood plasma, as does the brain cells and the muscle cells of our body
- That's why it's so important that our liver cells and kidney cells can properly regulate the levels of glucose in our blood

Q: What can tell us if gluconeogenesis or glycolysis predominates in the cell?
A: The
energy charge of the cell can tell us if gluconeogenesis or glycolysis
predominates

Q: What can tell liver and kidney cells if gluconeogenesis or glycolysis predominates in the cell?
A: Liver and kidney cells can also use the glucose levels in the blood to tell if glycolysis or gluconeogenesis predominate

Q: What must liver cells, and to a much lesser extent our kidney cells do to ensure proper glucose levels?
A: These cells must regulate glycolysis and gluconeogenesis in a reciprocal manner to ensure that proper glucose levels are maintained

Q: What is a key element to the reciprocal regulatory pathway of glycolysis and gluconeogenesis?
A: A key element in this reciprocal regulatory pathway is a bifunctional allosteric enzyme that contains 2 different regulatory domains
- One domain is called phosphofructokinase-2 (PFK-2), while the other regulatory domain is called fructose bisphosphate-2 (FBPase-2)

Q: Which 2 domains does the allosteric bifunctional enzyme contain?
A: One domain is called phosphofructokinase-2 (PFK-2), while the other regulatory domain is called fructose bisphosphate-2 (FBPase-2)

Q: In which 2 states does the bifunctional enzyme exist in?
A: - Unphosphorylated state
- Phosphorylated state

Q: What is the unphosphorylated state of the bifunctional enzyme?
A: In the unphosphorylated state, the PFK-2 domain is active while the FBPase-2 domain is inhibited

Q: What does the PFK-2 domain do?
A: The PKF-2 is responsible for converting fructose-6-phosphate into fructose-2,6-bisphospahte

Q: What is the phosphorylated state of the bifunctional enzyme?
A: In
the phosphorylated state, the PFK-2 domain is inactive while the FBPase-2
domain is active

Q: What does the FBPase-2 domain do?
A: FBPase-2 causes the dephosphorylation of fructose-2,6-bisphosphate into fructose-6-phosphate

Q: What happens to the PFK-2 domain in the phosphorylated state?
A: PFK-2 is inactivated and cannot stimulate fructose-2,6-bisphosphate production

Q: What else can affect the rate of gene transcription?
A: Insulin and glucagon can also affect the rate of gene transcription

Q: What does the influence of insulin do?
A: Insulin helps express proteins phosphofructokinase, pyruvate kinase and glucose transporters  (GLUT-4)

Q: What does the influence of glucagon do?
A: Glucagon on the other hand inhibits the expression of phosphofructokinase and pyruvate kinase, while stimulating the expression of fructose-1,6-bisphosphate and phosphoenolpyruvate carboxykinase