Chp 23: ATP from Fatty Acids

Question 1

1. Between meals, lipolysis is activated in adipose tissue as a result of changes in hormone concentrations. Which responsible hormones are increased or decreased?

Answer 1

• In the fasting state, insulin goes down and glucagon goes up. Decrease in insulin and increase in glucagon are responsible for release of fatty acid in adipose tissue
• Other info: Because there is so much fat stored, fatty acids are released from the adipose tissues long after glycogen stores are depleted in the liver. There is a limited amount of glycogen stored in the liver, with slightly more stored in the muscles
• Lipolysis is also increased by high epinephrine and cortisol. These hormones can be activated as the result of stress and exercise. The effects of all the stress hormones (epinephrine, glucagon, and cortisol) become more pronounced the longer a fasting state continues
• Between meals, there is a decrease in insulin levels and an increase in glucagon. The longer the fast continues, the more stress hormones rise

Question 2

2. How are free fatty acids transported from adipose tissue to muscle or liver cells?

Answer 2

Fatty acids diffuse from adipose cells and are transported by serum albumin. They travel in blood, bound in the hydrophobic binding pocket of albumin. They enter the cell via a fatty acid binding protein in the plasma membrane and are transported within the cell by another fatty acid binding protein. Fatty acids cannot travel in solution because they are hydrophobic

Question 3

3. Name the major pathway used to oxidize fatty acids into acetyl CoA.

Answer 3

• Acetyl CoA is produced from oxidation of fatty acids in the pathway of beta-oxidation
• Note: Before entering the beta-oxidation pathway, the fatty acid is activated to fatty acyl CoA by fatty acyl CoA synthetase

Question 4

4. What are the reactants and products of the fatty acyl CoA synthetase reaction?

Answer 4

Fig 23.2

• Fatty acyl CoA synthetase (fatty acid activation):
• ATP + fatty acid + CoA → fatty acyl CoA + AMP + PPi
• This reaction is endergonic and will not be spontaneous
• In the cells, inorganic phosphatase hydrolyzes the high energy bond of PPi to make the two linked reactions exergonic:

ATP + fatty acid + CoA → fatty acyl CoA + AMP + PPi (endergonic)

PPi  2 Pi (exergonic)_____________________________________

ATP + fatty acid + CoA → fatty acyl CoA + AMP + 2 Pi (exergonic

• Both high energy bonds in ATP have been hydrolyzed in order to provide the energy for the synthesis of the acyl CoA bond
• When calculating the energy derived from beta-oxidation, this 2nd ATP bond must be included in the calculations
• Other info: PPi is called pyrophosphate – P represents phosphate. P attached to an organic compound is organic phosphate (R-P), P alone is inorganic phosphate free in solution
• Fatty acid activation is the creation of a fatty acyl CoA from a fatty acid and ATP

Question 5

5. Describe the pathway for transport of fatty acyl CoA in the cytosol to fatty acyl CoA in the mitochondria. Use the terms carnitinepalmitoyltransferase I and II, carnitine, CoA, inner mitochondrial membrane, and carnitine acylcarnitine translocase in your explanation.

Answer 5

Fig 23.5

• Step 1: CPT 1 (carnitine palmitoyltransferase I) in the outer mitochondrial membrane transfers the activated fatty acyl group from fatty acyl CoA to carnitine and releases CoASH
• Step 2: Carnitine acylcarnitine translocase is an antiporter that transports fatty acyl carnitine into the matrix in exchange for carnitine
• Step 3: CPT II (carnitine palmitoyltransferase II) in the matrix transfers the activated acyl group from fatty acyl carnitine to CoA and release carnitine

Other info: Fatty acyl CoA in the matrix is now available for beta-oxidation. The entire pathway is reversible despite the way the arrows are drawn in the figure. CoA and CoASH both represent the same coenzyme A. The outer mitochondrial membrane has pores that easily let compounds of molecular weight less than 700 cross the outer membrane into the intermembrane space. The fatty acyl CoA has no trouble diffusing to the CPT on the inside of the outer mitochondrial membrane

Question 6

6. What are the names for the B-oxidation pathway?

Answer 6

Beta-oxidation

Question 7

6. What are the functions of B-oxidation?

Answer 7

Beta-oxidation of fatty acyl CoA

Question 8

6. What are the substrates of B-oxidation?

Answer 8

• fatty acyl CoA
• CoASH
• FAD
• NAD+

Question 9

6. What are the products of B-oxidation?

Answer 9

• acetyl CoA
• FAD(2H)
• NADH

Question 10

6. What is the control enzyme of B-oxidation?

Answer 10

acyl CoA dehydrogenase*

*This pathway is mainly controlled by the rate of release of fatty acids from adipose tissue and the fate of entry of faty acyl CoA into the matrix of the mitochondria. It is controlled to a lesser extent by the ratio of NADH to NAD+. However, even very high ratios will not totally inhibit beta-oxidation, just slow it down

Question 11

6. What is the regulation of B-oxidation?

Answer 11

Availability of fatty acyl CoA and NADH

Question 12

6. What are the compartments for B-oxidation?

Answer 12

mitochondria

Question 13

6. What are the tissues of interest in B-oxidation?

Answer 13

Every cell that has a mitochondria

Question 14

7. Given a saturated, straight chain fatty acid, calculate the number of molecules of Acetyl-CoA, FADH2, and NADH produced by B-oxidation.

How much ATP would this be equivalent to?

Answer 14

Fig 23.7

• The number of acetyl CoA molecules produced from a given saturated, straight chain fatty acid is determined by dividing the length of the chain in half: a 16-carbon chain produces 8 acetyl CoA molecules
• In order to convert the fatty acyl CoA into acetyl CoA, the beta oxidation cycle runs (N/2)-1 times. So in the previous example, it would be (16/2)-1 = 7 times. Each time that the cycle is run, it produces 1 FAD(2H) and 1 NADH and 1 acetyl CoA, except for the last time when it produces 2 acetyl CoA. Complete beta-oxidation of a 16 carbon fatty acid would therefore produce 8 acetyl CoA, 7 NADH, and 7 FAD(2H)
• Since each NADH is equivalent to 2.5 ATPs and each FAD(2H) is equivalent to 1.5 ATPs, complete beta-oxidation of a 16 carbon fatty acid would yield 28 ATPs
• Beta-oxidation does not include the oxidation of acetyl CoA by the TCA cycle. The TCA cycle is a separate pathway. When acetyl CoA is oxidized by the TCA cycle, 10 ATPs are produced by each acetyl CoA
• Remember, if it starts out as a fatty acid, not fatty acyl CoA, you must subtract 2 ATP from the total. This is ATP that is required to activate the fatty acid

Example 1: How many acetyl CoAs are produced by the beta-oxidation of a fatty acyl CoA containing 4 carbon atoms? How many ATP equivalents?

• 4 carbons = 2 acetyl CoA = one time through cycle = 4 ATP

Example 2: How many acetyl CoAs are produced by the beta-oxidation of a fatty acyl CoA containing 18 carbon atoms? How many ATP equivalents?

• 18 carbons = 9 acetyl CoA = 8 times through cycle = 32 ATP

Example 3: How many acetyl CoAs are produced by the beta-oxidation of a fatty acyl containing 18 carbon atoms? How many ATP equivalents?

• 18 carbons = 9 acetyl CoA = 8 times through cycle = 32 ATP – 2 ATP (since you needed 2 ATPs to activate the fatty acid) = 30 net ATPs

Question 15

8. Be able to name the three metabolites and two important cofactors in the conversion of part of an odd chain fatty acid to a TCA cycle intermediate.

Answer 15

Fig 23.11 - skip the epimerase reaction

Odd-chain fatty acids are also able to undergo beta-oxidation but differ from even-chain fatty acids in the last spiral. In this spiral 5 carbons remain – cleavage of the 5-carbon yields one more acetyl CoA and propionyl CoA. Propionyl CoA can be converted to the TCA cycle intermediate succinyl CoA and requires the following metabolites and cofactors to do so:

• Metabolites: propionyl CoA, methymalonyl CoA, and succinyl CoA
• Cofactors: B12 and biotin (add CO₂)

This is one of the anaplerotic pathways for the TCA cycle

Question 16

9. What are the major factors that control the synthesis of acetyl-CoA by B-oxidation in muscle and/or liver?

Answer 16

Fig 23.12

• As mentioned in the lecture, there are three points of control with many activators and inhibitors
• The release of fatty acids from adipose tissue. This controls the amount of free fatty acids in the cells of the body and how fast they can become fatty acyl CoA. The release from adipose is inhibited by insulin and stimulated by glucagon, epinephrine, and cortisol
• The second control occurs at CPT 1 (carnitine palmitoyltransferase 1). This is the enzyme that transfer the fatty acyl group from fatty acyl CoA to carnitine. CPT 1 is inhibited by malonyl CoA so when malonyl CoA ispresent, the fatty acyl group cannot be transferred to carnitine for entrance into the mitochondria. To run beta-oxidation, the concentration of malonyl CoA must be lowered
• Now, the concentration of malonyl CoA depends upon the activity of acetyl CoA carboxylase and this enzyme has several activators and inhibitors:
  
  • ATP/ADP&AMP ratio: When the cell needs energy, this ratio is low. AMP binds AMP-activated protein kinase. This kinase inhibits acetyl CoA carboxylase by phosphorylation. This lowers the concentration of malonyl CoA and activates CPT 1.
  • Glucagon and epinephrine activate protein kinase A that has the same mechanism as AMP-activated protein kinase (not shown in text)
  • Insulin causes activation of acetyl CoA carboxylase by dephosphorylation, the production of malonyl, and the inhibition of CPT 1
• Rate of ATP utilization by the ETC. if the ATP/ADP ratio is high, NADH and FAD(2H) will be in excess. Excess NADH and FAD(2H) will inhibit beta-oxidation but not totally. Under cellular conditions, high NADH and FAD(2H) slows beta oxidation but doesn’t stop it

Question 17

10. Name the substrate in the pathway for the synthesis of ketone bodies.

Answer 17

acetyl CoA

Question 18

10. Name the first ketone body made in the pathway for the synthesis of ketone bodies.

Answer 18

Acetoacetate

• Acetoacetate can then become either acetone via a spontaneous reaction, or beta-hydroxybutyrate: Acetoacetate + NADH + H⁺ ⇔ betahydroxybutyrate + NAD⁺
• 3 acetyl CoA are used to make HMG CoA, but one of those is subsequently released with HMG CoA lyase
• Any time there are more acetyl CoAs being produced than the mitochrondria can use, the liver will make ketone bodies and secrete them into the blood. This is a healthy reaction. Only with type 1 diabetes do you usually have enough ketone bodies to cause ketoacidosis. (Remember that ketone bodies were named before their structures were known – that is why beta-hydroxybutyrate, an alcohol, is called a ketone)

Question 19

10. What are the next two ketone bodies made in the pathway?

Answer 19

Acetone

Beta-hydroxybutyrate

Question 20

10. What is the intermediate that can be used either for ketone body synthesis or cholesterol synthesis?

Answer 20

3-Hydroxy-3-Methylglutaryl (HMG) CoA

Question 21

10. What is the enzyme that actually produces the first ketone body as a product?

Answer 21

HMG CoA-Lyase (Hydroxymethylglutaryl-CoA Lyase)

Question 22

10. Where does the pathway for ketone synthesis occur?

Answer 22

Mitochondria in the liver

Question 23

10. What is the control for the synthesis of ketone bodies?

Answer 23

Acetyl CoA concentration (excess lends itself to ketone body synthesis)

Question 24

11. Name a few tissues that oxidize ketone bodies. Why not the liver?

Answer 24

Skeletal muscle, heart muscle, brain, certain kidney cells, intestinal mucosa, and many other cell types use ketone bodies for energy production

Question 25

11. Why doesn’t the liver oxidize ketone bodies?

Answer 25

Liver makes ketone bodies, but does not use them because it lacks sufficient quantitities of the enzyme succinyl CoA and acetoacetate CoA transferase

Question 26

11. What happens to blood ketone bodies?

Question 27

11. Name the intermediates in the pathway from B-Hydroxybutyrate to acetyl CoA.

Answer 27

Cells transport both acetoacetate and beta-hydroxybutyrate from circulating blood into the cytosol and into the mitochondrial matrix. In the matrix, beta-hydroxybutyrate is oxidized to acetoacetate. CoA is transferred froms uccinyl CoA to acetoacetate, producing acetoacetyl CoA. The acetoacetyl CoA reacts with another molecule of CoA and becomes two acetyl CoAs. The acetyl CoA usually enters the TCA cycle to produce energy.

Question 28

11. What does the enzyme succinyl CoA:acetoacetate CoA transferase do?

Answer 28

Succinyl CoA: acetoacetate CoA transferase transfers CoA from succinyl CoA to acetoacetate to make acetoacetyl CoA

Question 29

12. What is the effect of insulin, glucagon, or epinephrine upon lipolysis in adipose tissue?

Answer 29

• Insulin inhibits lipolysis in adipose tissue during the fed state. About 2 hours after eating, insulin falls and glucagon rises. Glucagon (and epinephrine) stimulates lipolysis in adipose tissue as well as the subsequent release of fatty acids into the blood

Other info: In lipolysis, glucagon and epinephrine both activate the cAMP cascade and protein kinase A. PKA phosphorylates and activates hormone sensitive lipase (HSL). This enzyme hydrolyzes fatty acids from adipose triacylglycerols so that they can be released into the blood. HSL is phosphorylated/activated by PKA when cAMP levels are elevated. Therefore, lipolysis is active when levels of glucagon and epinephrine are high, likei n the fasting state and during exercise.

• Insulin, however, stimulates the phosphatase, which dephosphorylates and inactivates HSL. So in the fed state, lipolysis is inhibited by high levels of insulin
• Adipose cells do not perform beta-oxidation – if there is no glucose available, they use ketone bodies for fuel

Question 30

13. What happens to the blood levels of fatty acids, glucose, and ketone bodies during an extended fast? Explain how the use of ketone bodies by the brain spares muscle protein.

Answer 30

Fig 23.20

• Fatty acids increase for 2-3 days and then stay constant throughout starvation
• Blood glucose is maintained, but at the low end of normal (due to gluconeogenesis)
• Ketone bodies (acetoacetate steadily and beta-hydroxybutyrate) increase for 20-30 days. After 3 days, the brain uses more and more ketone bodies and less and less glucose to meet its energy needs
• When the brain uses less glucose during a prolonged fast (starvation), less glucose has to be made by gluconeogenesis in the liver. Gluconeogenesis uses amino acids that come from muscle as a substrate. If the liver uses less amino acids, less muscle protein will have to be broken down to provide the amino acid. Thus ketone bodies spare muscle protein

Question 31

14. If a person eats a balanced meal, does not exercise, and then begins a 10 hour fast, what happens to the rate of carbohydrate and fatty acid oxidation in muscle? Assume that the person does not exercise. What would happen if they began to exercise vigorously after 5 hours?

Answer 31

• During the fed state with insulin high and glucagon low, the use of carbohydrate (glucose) by muscle is high. Glycolysis and glycogen synthesis are both activated. During this time, the resting muscle is getting around 2/3 of its total energy from glucose and 1/3 from fatty acids
• As digestion is completed (2-3 hours), blood glucose decreases, insulin decreases, and glucagon increases. As a result, much less glucose will be used to make glycogen and to run glycolysis. Also, fatty acid mobilization from adipose will be greatly activated. The muscle will not get 2/3 of its energy from fatty acids and less than 1/3 from glucose
• If vigorous exercise begins, muscle activity and epinephrine will increase greatly. Fatty acid utilization by muscle will increase but there is a limit to the rate at which fatty acids can be transported to the liver and converted to acetyl CoA. The liver will use all the fatty acid it can get, but it won’t be near enough to meet its energy demands
• Epinephrine will increase glycogen breakdown, muscle glycolysis, and the uptake of glucose from the blood. With increasing exercise, glucose utilization will be greater than fatty acid utilization. The amount of glucose used by oxidative phosphorylation will be limited by oxygen and the ATP/ADP&AMP ratio will still be low. This will keep anaerobic glycolysis active all during the vigorous exercise

Question 32

15. How can a decrease in the insulin/glucagon ratio explain the increased production of ketone bodies during a fast?

Answer 32

• Once a fast begins, the level of glucose in the blood will drop. Since blood glucose is a major determinant of blood insulin and glucagon, the insulin to glucagon ratio will drop. The longer the fast lasts, the more epinephrine is released, which will decrease the insulin:glucagon ratio even further
• Insulin inhibits the release of fatty acids from adipose, and both glucagon and epinephrine activate fatty acid release from adipose. Fatty acids release will be high, so the concentration of fatty acids in the blood and liver cells will be high
• Insulin activates while glucagon and epinephrine inhibit acetyl CoA carboxylase. The concentration of malonyl CoA will be very low so carnitine palmitoyltransferase will be very active. The rate of fatty acyl coA entering the mitochondria and going beta-oxidation will be very high. Acetyl CoA concentrations will be very high. Whenever more acetyl CoA is made than can be oxidized by the TCA cycle, ketone bodies are made
• This is a mechanism for shipping to other cells the energy that the liver cannot use

Question 33

16. Concerning Otto shape, what hormonal changes occur during the long distance run and how do they affect the release of free fatty acids from adipose tissue?

Answer 33

• When Otto starts to run, he decreases the ATP to ADP&AMP ratio in his muscle cells, releasing epinephrine from his adrenal glands. Both result in more glucose entering muscle cells and the activation of glycolysis. The lower blood sugar and epinephrine both lower blood insulin. As he becomes more stressed during the run, cortisol will increase along with epinephrine and glucagon
• Cortisol induces enzymes for fatty acid release in adipose. Glucagon and epinephrine both activate hormone sensitive lipase (HSL). The inhibition of insulin upon HSL is removed. All of these contribute to increased release of fatty acids from adipose tissue

Question 34

17. Concerning Otto shape, during his long distance run the change in the concentration of AMP ensures the increased uptake of fatty acyl CoA into his muscle mitochondria.

Answer 34

• Muscle contraction requires the hydrolysis of a large quantity of ATP into ADP
• Increased ADP greatly increases the level of AMP
• Increased AMP activates AMP-dependent protein kinase
• AMP protein kinase phosphorylates and inhibits acetyl CoA carboxylase
• The concentration of malonyl CoA decreases so carnitine palmitoyltransferase becomes more active
• More fatty acylcarnitine is formed and enters the mitochondria through carnitine-acylcarnitine translocase
• More fatty acyl CoA is formed in the mitochondria

Question 35

18. Concerning Otto shape, during his long distance run the change in the concentration of AMP ensures the increased uptake of glucose into muscle tissue. How does this happen?

Answer 35

• As Otto’s muscles contract, ATP is used to generate the movement. As ATP decreases, ADP&AMP increase. The increased AMP activates AMP-dependent protein kinase (AMPK). Besides inhibiting acetyl CoA carboxylase, AMPK increases the number of glucose transporters in the muscle membrane

Other info: The major transporters of glucose across the cell membranes of muscle and adipose cells is an isozyme called glucose transporter IV (GLUT4). In a resting and exercising muscle cell, glucose entry is limited by the number of glucose transporters in the membrane. Both insulin and AMP-dependent protein kinase cause these transporters to move from vesicles inside the cell into the cell membrane.

• In the resting state, GLUT4 is largely under the control of insulin. In the exercise state, GLUT4 is under the control of AMPK. Also, since glycolysis is very active in the exercise state, the glucose entering the cell is rapidly metabolized
• Other cell types have other isozymes of glucose transporters that are not under the control of insulin or AMPK

Question 36

19. Concerning Otto shape, during his long distance run the change in the concentration of ADP causes increased B-oxidation.

Answer 36

• • During beta-oxidation there is one reaction that uses NAD+ as a substrate and produces NADH + H+ as a product. Raising the concentration of NAD+ or lowering the NADH + H+ will make the free energy change of the reaction more negative, and the reaction will speed up
• Likewise, during B-oxidation there is one reaction that uses FAD as a substrate and produced FAD(2H) as a product. Raising the concentration of FAD or lowering the FAD(2H) will make the free energy change of the reaction more negative, and the reaction will speed up
• Increasing the concentration of ADP in the muscle cell speeds up the ATP synthase reaction, lowering the proton gradient and speeding up the ETC. This will oxidize more NADH + H+ and FAD(2H), raising the concentration of NAD+ and FAD. As noted above, this increases the rate of beta-oxidation

Note! The two more important points of regulation of fatty acid oxidation are the release from adipose cells and the activation of the CPTI reaction. The control of beta-oxidation by the NAD/NADH ratio and FAD/FAD(2H) is not as important

Question 37

20. Concerning Lofata Burne: Explain why medium chain acyl CoA (MCAD) deficiency would cause a decrease in ketone body synthesis during a fast.

Also, from an energy point of view, explain why MCAD deficiency would increase the utilization of blood glucose by most tissues of the body and why gluconeogenesis in the liver is less than expected.

Answer 37

• Medium chain acyl CoA Dehydrogenase Deficiency (MCADD) results in high fatty acid concentration in blood but not the expected high concentration of ketone bodies during the fasting state
• She is not generating acetyl CoA from beta-oxidation of fatty acids due to MCADD. Long chain fatty acids are broken down to medium chain length (C6-C12), but she lacks the enzymes to further oxidize these medium length fatty acyl CoAs to acetyl CoA. The pathway backs up and even goes in reverse. The concentration of medium chain & long chain acyl CoA and carnitine all increase in the mitochondria and in the cytosol. They even show up as soluble acylcarnitines in the blood
• Ketone body synthesis depends upon more acetyl CoA being made than can be used by the TCA cycle. This requires a very active mobilization and utilization of fatty acids. In the case of MCADD, beta-oxidation is barely running or stopped. The concentration of acetyl CoA will not be high enough to produce ketone bodies
• Most tissues that depend on fatty acid oxidation for energy needs now have to get energy from glucose. When acetyl CoA cannot be made from fatty acids, they must rely on the oxidation of glucose or amino acids (protein stores). Glycolysis will be fully activated in most non-liver tissues and glycogenolysis will be fully active in liver. The body will rapidly run out of glucose and available glycogen
• Gluconeogenesis in the liver takes ATP and without beta-oxidation, there is not enough ATP. So glucose cannot be provided to the tissues via gluconeogenesis

Other info: The first step in beta-oxidation is actually catalyzed by a set of isozymes that have a preference for different chain lengths in fatty acids. Very long chain, long chain, medium chain, and short chain acyl CoA dehydrogenases are common in the mitochrondria

Question 38

21. Concerning Di Abietes who suffers from Type I diabetes, what is the cause of her disease?

Answer 38

Type 1 diabetes is an absolute inability of the beta cells of the pancreas to secrete insulin. This is an autoimmune disease

Question 39

21. Concerning Di Abietes, what effect does her Type I diabetes have upon blood concentrations of glucagon, catecholamines, and cortisol?

Answer 39

Without the presence of insulin, glucagon levels increase dramatically (since insulin inhibits glucagon secretion). Catecholamines and cortisol rise in the blood as a physiological response to the stress of a disease

Question 40

21. Concerning Di Abietes, what effect do the hormones glucagon, catecholamines, and cortisol have upon fatty acid mobilization from adipose tissue?

Answer 40

These hormones increase concentrations of the above hormones stimulate lipolysis and the mobilization of fatty acids from adipose tissue

Question 41

21. Concerning Di Abietes, what effect does low insulin and high glucagon have upon fatty acyl CoA entrance into liver mitochondria?

Answer 41

Furthermore, the absence of insulin limits the allosteric activation of acetyl CoA carboxylase, so the concentration of malonyl falls. Also, low insulin increases the release of glucagon and the production of active protein kinase A. Protein kinase phosphorylates and inhibits acetyl CoA carboxylase, lowering the concentration of malonyl CoA. Lower malonyl CoA removes the inhibition from CPT-1 and there is an increase of fatty acyl CoA into the mitochondria

Question 42

21. Concerning Di Abietes, what is the effect upon B-oxidation?

Answer 42

The rate of beta-oxidation is largely controlled by substrate availability. Since the concentration of fatty acyl CoA is greatly increased, the rate of beta-oxidation and production of acetyl CoA is greatly increased

Question 43

21. Concerning Di Abietes, what is the effect upon ketone body synthesis?

Answer 43

As acetyl CoA concentration builds up in the mitochondria, ketone body synthesis increases. The more acetyl CoA, the more ketone bodies are synthesized and transported to the blood.

Question 44

21. Concerning Di Abietes, what is the effect upon blood pH?

Answer 44

Since acetoacetate and beta-hydroxybutyrate are acidic, overproduction will cause metabolic acidosis (ketosis)