Glycogen metabolism, glycolysis

Question 1

In what state is glucagon expressed? By what general mechanism does it act?

Answer 1

In the unfed state.

-acts by increasing cAMP in liver and adipose to make energy (catabolism) and decrease glycolysis.

Question 2

In what state is epinephrine expressed? By what general mechanism does it act?

Answer 2

Fight or flight

-acts by increasing cAMP and calcium intake in muscle, adipose, and liver (muscle contraction!) to increase energy production by catabolism, including glycolysis

Question 3

In what state is insulin expressed? By what general mechanism does it act?

Answer 3

Fed state

-increases tyrosine kinase activation and PIP3 in muscle, adipose, and liver, as well as inhibition of cAMP to increase uptake of glucose and glycogen synthesis while preventing energy formation.

Question 4

In general, how is metabolism coordinated?

Answer 4

Hormone signaling

Question 5

What is the insulin agonist?

Answer 5

epinephrin or glucagon

Question 6

What receptor does epinephrine and glucagon bind to? What is the effect?

Answer 6

Glucagon and epinephrine can bind to GPCR. Effect is activation of adenylate cyclase –> cAMP –> protein kinase A –> kinase activity. Epinephrine can also elicit IP3 formation from PIP3 using phospholipase C, activated by G subunit alpha.

Question 7

What effects do epinphrine and insulin have on PIP3?

Answer 7

Epinephrine cleaves it to form IP3, insulin phosphorylates PIP2 to form PIP3.

Question 8

Which GLUT transporter is insuline dependent?

Answer 8

GLUT4 in muscle and adipose. GLUT4 transporter is endocytosed into the cytoplasm in the absence of insulin, and added back to the membrane when insulin is present.

Question 9

Describe the Km values for the GLUT transporters, and how they relate to normal circulating glucose concentrations.

Answer 9

The normal circulating glucose level is 5mM. 3 of the GLUT transporters have Km values around that. These transporters rely mostly on glucose. GLUT2 in the liver, however, has a much higher Km, allowing glucose uptake only after a meal so that at normal or unfed conditions, it can send glucose off for other tissues such as the brain to use.

Question 10

Describe an experiment supporting the insulin dependence of GLUT4.

Answer 10

A GLUT4 protein is MYC-tagged in the extracellular space, and EGFP-tagged on the cytoplasm side. We see basal GFP in the absence of insulin, and no MYC antibody is detected. When insulin is added, the MYC antibody signal overlaps with the GFP, showing the protein has moved to the cell membrane.

Question 11

If glucose transport into and out of the cell is by diffusion, how do we regulate glucose in the cell?

Answer 11

It is phosphorylated at carbon 6 by one of two isoforms. Glucokinase in the liver, or hexokinase everywhere else. Glucose-6-P cannot pass through the membrane like glucose.

Question 12

Describe the differences between glucokinase and hexokinase.

Answer 12

Glucokinase (liver) has a high Km for glucose, so it does not grab glucose as readily as hexokinase. Hexokinase (all other tissues) has a low Km for glucose, so it phosphorylates it more readily. Hexokinase is also inhibited by its product, glucose-6-P.

Question 13

Describe the kinetics of glucokinase.

Answer 13

It is sigmoidal, suggesting allostery. The inflection point is around normal glucose levels so that glucokinase can be more sensitive. If glucose decreases a little, glucokinase activity decreases a lot. If glucose inreases a little, glucokinase activity increases a lot.

Question 14

Describe how the monomeric protein glucokinase can be regulated by allostery.

Answer 14

If glucose binds, it falls into a closed conformation and becomes active. When ATP binds it is locked in. When glucose is not plentiful, glucokinase falls into an open and inactive conformation, which is slow to getting back to its open form.

Question 15

Which types of tissues are glucose-dependent?

Answer 15

RBC, brain, renal medulla (i.e. these use only glucose for energy production). These tissues need glucose whether is it available or not, so glucose transport is insulin-independent.

Question 16

Which tissues are glucose-independent?

Answer 16

Muscle, heart, adipose (though glucose transport is insulin-dependent). These tissues can store glucose.

Question 17

Which tissues are glucose-producing?

Answer 17

Liver. Production is insulin-independent

Question 18

Glucose phosphorylation by hexokinase is controlled by what type of regulation?

Answer 18

allosteric regulation by the product

Question 19

Glucose phosphorylation by glucokinase is controlled by what type of regulation?

Answer 19

Substrate concentration.

Question 20

What different pathways can glucose-6-P follow?

Answer 20

• glycolysis
• hexose monophosphate pathway
• glycogen formation

Question 21

What types of bonds are found in glycogen?

Answer 21

• between linear glucose monomers: alpha 1,4 linkage
• between branch point: alpha 1,6 linkage

Question 22

Why is excess glucose not just stored as glucose?

Answer 22

Each individual glucose monomer draws in water by osmosis. Osmosis looks at the number of individual molecules, not the size of each molecule, so by forming a single molecule of glycogen (albeit a large molecule), it can avoid osmotic pressure.

Question 23

What is the function of glycogen in the liver versus in the muscle?

Answer 23

Liver: maintain blood glucose levels for all tissues

Muscle: fuel reserve for its own ATP generation

Question 24

What are the three sources of glucose?

Answer 24

diet - immediate

glycogen - hours

gluconeogenesis - days

Question 25

Which tissue has the ability to dephosphorylate glucose-6-P with a phosphatase?

Answer 25

The liver, so it can mobilize glucose to other tissues in need.

Question 26

Describe the process of glycogen synthesis.

Answer 26

1. glucose-6-P is isomerized by phosphoglucomutase to glucose-1-P 2. glucose-1-P is activated by UDP-glucose phosphorylase using UTP to produce UDP-glucose and pyrophospate (PPi). PPi is hydrolyzed to inorganic phosphate, which drives the reaction forward. 3. nascent glycogen chain is primed, as glycogen synthase cannot start from scratch, by UDP-glucose onto glycogenin, leaving behind UDP. UDP-glucose can be added to this by glycogen synthase. Thus, at the core of every glycogen granule is a glycogenin molecule. 4. Glycogen is branched by the branching enzyme, by taking a chunk from the linear chain and moving it to a branching point.

Question 27

Why is glycogen branched?

Answer 27

Phosphorylase chews away at glycogen from the end of the chain one molecule at a time. Thus, if there are more branches, then glucose can be mobilized more quickly if and when it is needed.

Question 28

Why do plants store glucose in amylose in a linear chain witout any branches?

Answer 28

It does not need to rapidly mobilize glucose as we do.

Question 29

Describe the breakdown of glycogen.

Answer 29

1. glycogen phosphorylase shortens linear chains one molecule at a time by inserting a phosphate between bonds, forming glucose-1-P. (saves energy so we don't have to re-phosphorylate glucose for use in glycolysis) 2. phosphoglucomutase converts glucose-1-P to glucose-6-P for use in glycolysis or hexose pathway. 3. debranching enzyme cuts off most of the branch and adds to the end of a linear chain for degradation by glycogen phosphorylase, and then a single glucose left behind is hydrolyzed to produce glucose (not glucose-1-P!)

Question 30

How does glycogen phosphorylase overcome the competition of hydrolysis to break glycogen chains?

Answer 30

It holds glycogen very tightly so as to exclude water.

Question 31

How much glucose is produced per breakdown of a glycogen branch?

Answer 31

1 molecule of glucose per branchpoint.

Question 32

Describe the energetics of glycogen synthesis.

Answer 32

1. energy input to activate glucose-1-P with UDP 2. ATP to regenerate UTP Total: 1 ATP molecule used.

Question 33

Describe the energetics of glycogen breakdown.

Answer 33

1. input of one inorganic phosphate per glycosidic bond broken via phosphorolysis 2. one ATP molecule needed for every removal of a glucose at a branch point

Question 34

Describe the net energetic cost of using glycogen to store energy.

Answer 34

Building up: 1 ATP per glucose molecule Breaking down: 1 ATP per branch point **Total:** if a branch occurs every ~10 glucose monomers, totally energy to store glycogen is ~1.1 ATP per glucose molecule (4% storage cost compared to ATP afforded by each glucose molecule)

Question 35

Describe very generally the regulation of glycogen storage.

Answer 35

Reciprocal regulation: synthesis and degradation cannot occur at the same time. If glycogen is being made, it cannot be broken down.

Question 36

Which two enzymes involved in glycogen storage are regulated?

Answer 36

- glycogen phosphorylase (breakdown) - glycogen synthase (synthesis)

Question 37

Describe the regulation of glycogen breakdown.

Answer 37

Regulation of glycogen phosphorylase - hormonal regulation: phosphorylated and activated in presence of glucagon - allosteric regulation: in muscle, low energy signal AMP activates phosphorylase. ATP and glucose-6-P inhibtis it. In liver, glucose inhibits phosphorylase. **Combined effects of global and local signals contribute to the final decision.**

Question 38

Describe how phosphorylation can alter the conformational equilibrium of an enzyme.

Answer 38

An enzyme is in conformational equilibrium normally. Phosphorylation does not turn on or off the activity of an enzyme, but rather shifts the conformational equilibrium in one direction or another --\> towards more active or less active.

Question 39

Describe in detail the homronal control of glycogen phosphorylase.

Answer 39

Glucagon: 1. Glucagon (elicits phosphorylation events) leads to phosphorylation and partial activation of phosphorylase kinase. 2. muscle contraction releases Ca2+ which further activates phosphorylase kinase 3. Now fully activated phosphorylase kinase phosphorylates glycogen phosphorylase, forming phosphorylase a. Insulin: 1. protein phosphatase 1 is phosphorylated and activated. 2. PP1 removes phosphate from glycogen phosphorylase, inactivating it. 3. phosphorylase kinase is also dephosphorylated by PP1, so it can not re-phosphorylate glycogen phosphorylase

Question 40

Describe in detail the hormonal regulation of glycogen synthase.

Answer 40

Glucagon regulation: 1. Protein kinase A is activated via phosphorylation 2. glycogen synthase is phosphorylated by PKA, inactivating it Insulin regulation: 1. Protein phosphatase 1 is phosphorylated and activated. 2. glycogen synthase is dephosphorylated by PP1, activating it 3. phosphorylase kinase is also dephosphorylated and inactivated by PP1, so it can no longer phosphorylate and inactivate glycogen synthase

Question 41

How can activation of protein phosphatase 1 by insulin be antagonized?

Answer 41

If glucagon is expressed, PKA will be activated. PKA can phosphorylate PP1 at a different, regulatory site than that which is phosphorylated to activate it, thus inactivating it.

Question 42

How do we know that glycolysis is the original energy-producing pathway?

Answer 42

It is found in all domains of life.

Question 43

Where do we see energy coupling in glycolysis? Describe the process.

Answer 43

Conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate by dehydrogenase. The aldehyde is oxidized to a carboxylic acid. Because addition of a phosphate to an acid is unfavorable, the aldehyde is not directly oxidized to an acid. 1. aldehyde forms hemithioacetal with thiol group on enzyme 2. NAD oxidizes hemithioacetal to thioester (more suceptible to nucleophilic attack that acid) 3. Phosphate can attack carbonyl carbon, releasing enzyme+thiol as leaving group. Thioester bond was high energy, causing this attack to be energetically favorable.

Question 44

Name the mechanisms by which redox balance is maintained between the cytosol and the mito (i.e. moving and freeing up electron shuttles).

Answer 44

1. malate transporter and aspartate transporter. 2. glycerol-3-phosphate shuttle. 3. pyruvate to O2 via ox phos 4. pyruvate to lactate in anaerobic muscle to regenerate NAD 5. pyruvate -\> acetaldehyde -\> reduced to ethanol to regenerate NAD

Question 45

Why is it important to be able to maintain a redox balance between the cytosol and mito?

Answer 45

NADH carrying electrons extracted from glucose must be able to get to mito to use them in the ETC. Oxidized NAD in the mito must be able to get back to the cytoplasm to reduce metabolites of glucose.

Question 46

Describe maintenance of the redox balance using malate/aspartate shuttle.

Answer 46

**In cytoplasm:** 1. aspartate is converted to OXA 2. OXA is reduced to malate using NADH, forming NAD 3. malate is transported into the mito **In mito:** 4. malate is oxidized to OXA, regenerating NADH 5. OXA is converted to aspartate, which moves to the cytoplasm via aspartate transporter

Question 47

What proves the importance of maintaining redox balance between cytosol and mito?

Answer 47

There are more than one pathway to maintain it.

Question 48

Describe redox balance maintenance via the glycerol-3-phosphate shuttle system.

Answer 48

**in cytoplasm:** 1. didroxyacetone is reduced to glycerol-3-phosphate using NADH 2. glycerol-3-P, still in cyto, is oxidized back to didroxyacetone glycerol 3-phosphate dehydrogenase, reducing FAD to FADH2. 3. FADH2 then reduces ubiquinone

Question 49

Describe Warburg effect.

Answer 49

In cancer cells, the majority of glucose is used for glycolysis even if O2 is available. This prevents the full breakdown of glucose to CO2 so that the carbon skeleton can be retained and used a building blocks. Therefore, NAD is recycled by reducing pyruvate to lactate.

Question 50

What are the three, regulated checkpoints of glycolysis?

Answer 50

1. hexokinase 2. phosphofructokinase 3. pyruvate kinase

Question 51

Describe the effect of ATP on PFK1 activity.

Answer 51

ATP creates a biphasic kinetic graph. Because ATP is a substrate for glycolysis, small increases when ATP is low increases velocity. However, at a certain point, increasing ATP levels decreases PFK1 velocity because ATP is no longer needed.

Question 52

Describe the negative allosteric effectors of PFK1.

Answer 52

1. ATP 2. 3-phosphoglycerate, PEP, citrate (glycolysis intermediates) 3. phosphocreatine (alternate energy source in muscle) 4. H+ and lactate (too much acid can poison the cell)

Question 53

Describe the positive allosteric effectors of PFK1.

Answer 53

1. AMP and ADP (low energy signals) 2. fructose-6-phosphate (substrate of PFK1) 3. fructose-2,6-diphosphate (derived from hormonal signals)

Question 54

Describe kinetic properties of PFK1 with changing concentrations of fructose-6-phosphate.

Answer 54

fructose-6-P is an allosteric effector, so the kinetic graph is sigmoidal.

Question 55

What is the point of fructose-2,6-diphosphate regulation of PFK1?

Answer 55

- make glycolysis sensitive to hormonal contorl - uses a 2nd kinase that recognizes fructose-6-phosphate

Question 56

Describe the regulation of PFK1 by fructose-2,6-bisphosphate and PFK2.

Answer 56

**Unfed state:** 1. glucagon signals activation of PKA. 2. PKA phosphorylates PFK2's kinase domain, inhibiting it and turning on its phosphatase domain. 3. PFK2 phosphatase removes phosphate from fructose-2,6-bisphosphate, forming fructore-6-phosphate. **Fed state:** 1. Phosphoprotein phosphatase removes phosphate group from PFK2 kinase domain, activating it. 2. PFK2 phosphorylates fructose-6-phosphate, forming fructose-2,6-phosphate (fructose-6-P positively effects phosphoprotein phosphatase) 3. fructose-2,6-bisphosphate stimulates PFK1, so fructose-1,6-bisphosphate can be formed and glycolysis can continue.

Question 57

What is the primary positive effector of PFK1 in liver, heart, muscle, and all tissues? Under what physiological conditions?

Answer 57

liver: fructose-2,6-bisphosphate (high blood sugar - glucagon signal) heart: fructose-2,6-bisphosphate (stress - epinephrine signal) muscle: AMP, fructose-2,6-bisphosphate (contraction) other tissues: AMP (anoxia - low O2)

Question 58

Describe pyruvate kinase regulation.

Answer 58

Pyruvate kinase is a homotetramer subject to alosteric (local) and covalent (global; phosphorylation) regulation. - equilibrium shifted to active state when phosphorylated - binding of PEP or fructose-1,6-bisphosphate shifts to active state - binding of ATP and alanine shifts the equilibrium towards the inactive state

Question 59

Describe the molecular structure of pyruvate kinase, and how it lends to regulation.

Answer 59

each monomer has: - phosphate binding site on back (tetramer conformational rearrangement at subunit interface allows phosphate binding to affect catalytic subunit activity) - catalytic subunit on front - allosteric binding site

Question 60

Describe the isoforms of pyruvate kinase and how they are differentially regulated.

Answer 60

4 isomers from 2 genes... **pyruvate kinase L gene:** -two potential start sites. primary transcripts either contain first or first and second start site. processing removes one of them. the L start site contains a phosphorylation site, but the R start site does not. thus, pyruvate kinase in some tissues cannot respond to hormonal signals **pyruvate kinase M gene:** -contains single start site, but C-terminal exons are edited differently depending on the tissue type.

Question 61

What is the purpose of the hexose monophosphate pathway?

Answer 61

To generate NADPH and building blocks for nucleic acids.

Question 62

What pulls the hexose monophosphate pathway forward?

Answer 62

- hydrolysis and opening of lactone - diffusion of CO2 out of the cell

Question 63

Where is the hexose monophosphate pathway regulated?

Answer 63

It is regulated at the first step, glucose-6-phosphate dehydrogenase, which forms the lactone from glucose-6-phosphate. It is regulated by the ratio of NADPH/NADP

Question 64

What is the final product of the h/exose monophosphate pathway?

Answer 64

ribose 5-phosphate

Question 65

What is the fate of extra ribose 5-phosphate produced by the hexose monophosphate pathway?

Answer 65

It is recycled into glycolytic intermediates. This happens if the cell is not dividing or getting ready to divide.

Question 66

What reaction types are the hexose monophosphate pathway divided into?

Answer 66

oxidative and non-oxidative reactions

Question 67

What is NADPH good for?

Answer 67

- biosynthetic reactions - protection from oxidative stress (in the cell, glutathione cysteine group is reversibly oxidized by ROS so more important molecules are not damaged. NADPH re-reduces glutathione so that it can continue to protect the cell from oxidative damage)

Question 68

What is the effect of a glucose 6-phosphate dehydrogenase deficiency?

Answer 68

This enzyme catalyzes the first step of the hexose monophosphate patwhay. Without it, NADPH will not be made and oxidized glutathione cannot be red-reduced. Causes breakage of the red blood cells (ROS's destroy the DNA), but is conserved in populations because it confers malaria resistance.

Question 69

What tissues is gluconeogenesis especially important for during periods of fasting?

Answer 69

The brain

Question 70

What about the three regulated steps of glycolysis must be considered when discussing GNG?

Answer 70

That because they are regulated, they cannot go backwards. Thus, GNG must use different reactions to bypass these steps.

Question 71

What is the first regulated step of GNG? How does it relate to glycolysis?

Answer 71

pyruvate conversion to PEP. This conversion requires two reactions: 1. pyruvate carboxylase uses ATP to convert pyruvate to OXA, and PEPCK uses GTP to decarboxylate OXA to PEP. This is also the last step of glycolysis, and is therefore pyruvate kinase is regulated in glycolysis.

Question 72

How much energy is required for the first step of GNG?

Answer 72

two ATP equivalents (one ATP and one GTP)

Question 73

What enzyme utilizes biotin?

Answer 73

pyruvate carboxylase uses it to convert pyruvate to OXA. biotin contains a CO2 binding region, which is used to carboxylate pyruvate to a 4-carbon molecule.

Question 74

What is the 2nd regulated step of GNG? How is it regulated?

Answer 74

Removal of phosphate from fructose-1,6-bisphosphate to form fructose-6-phosphate by fructose-1,6-bisphosphatase. It is inhibited by AMP, because energy is needed to put into GNG. It is also inhibited by fructose-2-6-bisphosphate (makes sense, as it stimulates opposite pathway of glycolysis) because it is generated in the fed state.

Question 75

What is the third regulated step of GNG? In what tissue does this take place?

Answer 75

It is the removal of phosphate from glucose-6-P by glucose-6-phosphatase (the "opposite" reaction as the first reaction of glycolysis). This really only takes place in the liver, because that is the only place where glucose needs to leave the cell.

Question 76

Describe regulation of the final GNG step.

Answer 76

Regulation of glucose-6-phosphatase. It is regulated by ER transporters. - glucose-6-P is in the cytosol, but glucose-6-phosphatase is in the lumen of the ER. - glucose-6-P must enter the ER lumen, through a trasnporter, be dephosphorylated, and leave the ER as glucose through a separate transporter

Question 77

Compare the energy consumption of ATP in glycolysis vs GNG.

Answer 77

- glycolysis nets 2 ATP - GNG uses 6 NTP. **therefore, because GNG is expensive, it is important that it is tightly regulated and not occurring at the same time as glycolysis**

Question 78

How is the first step of GNG regulated?

Answer 78

carboxylation of pyruvate to OXA can be stimulated by acetyl coA, a product of fat breakdown. This tells the cell thatif fat is being broken down, glucose needs to be produced. Acetyl coA also prevents the breakdown of pyruvate into acetyl coA by pyruvate dehydrogenase (product inhibition)

Question 79

Describe the reasoning behind alanine as an inhibitor of pyruvate kinase (last enzyme in glycolysis)

Answer 79

alanine is a building block that forms pyruvate in the breakdown of muscle. Thus, if muscle is being broken down to provide pyruvate for GNG, then glycolysis cannot be happening.

Question 80

Describe compartmentalization of GNG.

Answer 80

-first step takes place in mito, and OXA is shuttled as a reducing equivalent into the cytosol where it is decarboxylated into PEP.

Question 81

Describe a non-fasting application of GNG.

Answer 81

Because the RBC get energy only from glycolysis, and they regenerate NADH from lactate, they must somehow convert lactate back to RBC. However, they cannot do it alone. Lactate is transported from the RBC to the liver where it is converted to glucose, and then transported back to the RBC.

Question 82

Why is it energetically favorable to undergo GNG?

Answer 82

Although it costs 6NTP, glycolysis produces much more ATP.

Question 83

Other than pyruvate, what other carbon sources enter GNG?

Answer 83

lactate, amino acids, glycerol