FUNdamentals

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					FUNdamentals
Pritchard 11:00 – 12:00
09.04.08

Slide 1 : just outline slide
Slide 2
     The key is that glycogen is a rapidly mobilizable from of glucose. When you need glucose, you
         can get it from glycogen very fast.
     Brain needs 60% of glucose (different from earlier slide) and this changes with exercise, but the
         fact is that the brain needs glucose. The brain needs glucose to maintain the electrical potential
         of all the billions and billions of neurons in the brain, it has to be constantly cleaving, making
         ATP.
     To make ATP uses glucose, glucose breakdown. The brain does it oxidatively; you don’t do
         anaerobic glycolysis in the brain.
Slide 3
     Pointing out that there is glycogen stored both in the muscle and liver and skeletal muscle.
         Fairly high concentration in the muscle; doesn’t get as high as in the liver, but your mass of
         muscle is higher than your mass liver, so you have more muscle glycogen.
     Star this – muscle glycogen and liver glycogen serve different purposes. Muscle glycogen is
         storage form of quick energy for muscle movement; liver glycogen helps to maintain
         blood/glucose levels.
     We do maintain glucose levels constantly, unless you are diabetic. Normal people keep theirs at
         a milligram/mL most of the time, except for after a big meal or if you haven’t been eating.
     Glycogen metabolism is regulated at many levels. Interesting thing about glycogen stores is that
         they are not very big. Fat storage is weeks or months worth to supply energy; we could use all
         our glycogen up in an hour if we exercised very vigorously.
     The diagram illustrated the idea that glycogen levels go up and down during the day. After
         dinner high, sleeping low, after breakfast high again.
     Take home message of the slide: glycogen levels fluctuate during the day and go up when we
         eat.
Slide 4
     EM of a rat liver; one section (left) is when the rat was well fed; stained with a dye that shows
         glycogen granules in the liver.
     On the left there are lots of granules. If you starve the rat, even for a day, then you see there is
         hardly any glycogen granules.
     Point of the slide is to show that there are dramatic changes in glycogen over a relatively short
         time, like a day. If you vigorously exercise, can deplete glycogen in an hour.
Slide 5
     So why bother to store glycogen if it relatively a small amount as compared to fat? 3 reasons:
              o Can’t get energy out of fat as fast as you can get energy out of glycogen. (We will learn
                   about B-oxidation of fat later, and zipping it through the TCA to make ATP…) but you
                   can get ATP out of glycolysis very very fat. Glycogen supplies glucose6-P for glycolysis.
              o Fat can’t supply any energy in the absence of oxygen. The fat break down process in
                   called B-oxidation and that involves oxygen. Anaerobic glycolysis doesn’t require
                   oxygen (ie needing to make very fast movement over a relatively short period of time,
                   you are carrying out anaerobic glycolysis. That is another reason why you want to store
                   glycogen – to feed to make glycolysis. You get the glucose by breaking down glycogen.)

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     Fat cannot be converted to glucose and vital organs (brain) need glucose.
Slide 6
     Why not just store free glucose instead of making glycogen?
     If you are trying to fill up a cell with glucose, to shove in more glucose in that cell would take a
         fair amount of energy, requiring ATP to go against the concentration gradient. The whole goal is
         to use glucose to make ATP; don’t want to waste ATP getting glucose into a cell.
     Even if you could stuff the cell, you would get a high osmotic pressure within the cell and the cell
         would swell and pop.
     Talking about osmotic pressure, remember from 1st year chemistry that osmolarity is a
         colligative property. It depends on the number of partials and not how big they are.
     Freezing point depression – add salt or sugar – the freezing point gets lower than freezing. That
         is a colligative property too; it depends on the amount of particles and not what the substance
         is.
     Having loads of free glucose (hundred and thousands around) if you join the free glucose
         together to get a glycogen molecule, then you only have one particular and the osmotic
         pressure is very low.
     That is a way to avoid the problem of high osmotic pressure and concentration gradient.
Slide 7
     The final products of glycogen breakdown depend on muscle fiber. The nomenclature varies –
         sometimes see white muscle fibers referred to as fast twitch, and other as slow twitch, but
         people also just talk about red muscle fibers and white muscle fibers.
     In red muscle fibers, there are load of mitochondria and myoglobin, a heme containing protein.
         Glucose is primary converted to pyruvate in red muscle cells, and then it goes through pyruvate
         dehydrogenase converts it to acetyl CoA to enter the TCA and NAD and FADH from the TCA go
         through the ETC through oxidative phosphorlyation and you get a ton of energy out of the
         breakdown of glucose from glycogen from the red muscle fibers. Does this slowly.
     White muscle fibers have few mitochondria and very little myoglobin and they primarily break
         down glucose anaerobically, and the final product is lactatic acid.
              o They do that very fast.
Slide 8
     When you cook chicken breast, its white meat because it is filled with white muscle fibers.
     Cook a duck break and you see its dark – loads and loads of red muscle fibers. Flocks of geese,
         ducks – use breast muscle for flight.
     Don’t see chickens flying much – their rapid burst of energy (10)
     Turkeys don’t fly very far, so turkeys have white muscle fibers. They run a lot, so their legs have
         tons of red muscle fibers. Their legs are very dark meat.
     People – are a mix of white and red muscle fibers. About equal.
Slide 9
     Human muscle stained with 2 dyes differentially to show white from red; see that it is about
         50/50.
     Humans do short bursts of rapid movement and can do sustained movement.
Slide 10
     Lets look at the structure of glycogen. It should look familiar to you – looks like amylopectin
         that we looked at when we were talking about insoluble starch.
     Glycogen was once called animal starch.
     Looks like an amylase chain, a-1,4- glucose units. Pointed out starch is digestible and so is
         glycogen if you eat it in meat.

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       It is branched with a -1,6 branches. One different between glycogen and amylose is that
        glycogen is much more highly branched.
       Turns out when you have branch, you have an end. When you harvest glucose units from
        glycogen, you harvest them from the ends. Idea is that by having lots of branching you have lots
        of ends.
       Whole purpose of glycogen is to give an energy source of rapidly accessible glucose for energy
        that you might need in a few seconds.
       One branch for about every 10 sugars. 100,000 glucose units in one granule.
Slide 11
     Looking about glycogen biosynthesis - how it is made. Made in the cytoplasm in the cytosol.
     When you transfer glucose units or any other sugar, it is always the nucleotide sugar, and you
         have to activate that sugar.
     With glucose, that sugar is UDP. UDP glucose.
     Point out that UDP glucose is made from glucose-1-phosphate and UTP.
     Where does glucose-1-phosphate come from? G-6-P comes from eating glucose, gets into cells
         and it phosphorlyated.
     G-6-P can be readily converted to G-1-P by a phosphoglucomutase reaction. Mutase refers to
         an enzyme that transfers a group, like a phosphate, from one position to another.
Slide 12
     This is how you get G-1-P which reacts with UTP to get UDP-glucose. This is the form that
         transfers the glucoses when you are making glycogen.
Slide 13
     One of 2 really critical enzymes in glycogen metabolism. This is the enzyme that helps
         synthesize long chains of glycogen.
     UDP-glucose transfers a glucose to the end of a glycogen chain, lengthening it by one glucose.
         Its glycogen synthase reaction.
Slide 14
     Didn’t know this till 15 years ago – glycogen synthesis begins on a little protein called
         glycogenin.
     Glycogenin actually has catalytic activity; seeing a dimer of it here. The idea is that it catalyzes
         the addition of the first 7 glucose units.
     Dimer because you are supposed to imagined this one catalyzes the reaction here, the other
         one in the other spot. That may not be true.
     Main idea – 7 sugar units get added to the protein.
     Then glycogen synthase takes over and starts adding more units.
Slide 15
     Point out that the glycogen granule, these enzymes are not actually drifting about the cell freely
         in the cytoplasm. Different enzymes like phosphorylase kinase, glycogen phosphorylase and so
         are all associated weakly with the glycogen granule.
     Easy to forget, but that is what happens.
Slide 16
     Interesting thing – after the glycogen synthase extends the chain long enough, then an enzyme
         called glycogen branching enzyme takes over. It clips off about 7 glucose units from a long chain
         and transfers them to a position about 4 sugars away from the next branch. It generates a
         branch.



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        More importantly, it generates an end. And ends are where you harvest the glucose from. Then
         glycogen synthase makes it longer and longer and when it gets long enough, the glycogen
         branching enzyme does this again.
     It keeps moving 7 sugar chains, introducing more and more branches to get bigger.
    Slide 17
          When you break glycogen down, it’s not a hydrolysis – you don’t degrade glycogen into free
             glucose.
          It is a phosphorylysis. An enzyme, glycogen phosphorylase (generally see it referred to as
             phosphorlyase – but they mean glycogen phosphorylase). It does a phosphorolysis.
          See the inorganic phosphate? It cleaves right there and makes a G-1-P.
          Glycogen phosphorylase cleaves by introducing a phosphate. If it was a hydrolyase, it would
             cleave by introducing a water.
Slide 18
          Problem is that branches get in the way – you have to get rid of the branches. In the same
             way that there is a branching enzyme, there is a glycogen debranching enzyme.
          It does not transfer a 7 sugar unit; it clips off a piece that is 3 sugars long and transfers it to
             the end of a long chain, leaving a stump.
          See the same enzyme that catalyzes this transfer also cleaves the stump. The stump is
             cleaved off as free glucose.
          Remember, glycogen phosphorylase cleaves off a G-1-P where as this cleavage is a free
             glucose.
Slide 19
          What you end up with when you break down glycogen is 10x G-1-P as glucose.
          Again, what do you do with G-1-P? Told you already it can be converted to G-6-P by this
             enzyme.
          Slide shows how mutases work; mutases will invariably have an enzyme bound intermediate
             with this functional group on two positions. See what happens – you start in 1 postion and
             on the enzyme bound intermediate, you have another phosphate group added. Then this
             one is taken off, and this is released.
          That is how mutases works. Remember G-6-P can then go through glycolysis and make
             ATP.
          G-6-P is also the starting material in the pentose phosphate pathway (PPP.)
Slide 20
     Essential cofactor utilized in a pyridoxal phosphate reaction…
Q: What can G-6-P be used for?
A: Can funnel directly into glycolysis, or PPP or if this is in the liver or kidneys, you can cleave a
phosphate from G-6-P and dump the glucose into the bloodstream. Only kidney and liver have the
enzyme capable of taking off a phosphate.
     Not going to belabor this point…
     Pyridoxal phosphate is another one of those cofactors, vitamins and cofactors almost always.
         Vitamins are small molecules, usually essential cofactors for enzymes.
     Pyridoxal phosphate is used in all kinds of reactions. It is covalently bound by a Schiff base.
Slide 21
     Talk briefly about the regulation of glycogen synthesis. If you are fasting, you break down
         glycogen to get the glucose for energy.



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       You are not going to be making glycogen when you are fasting; on the other hand, if you have a
        good diet, you are going to starting down regulating glycogen. (don’t’ need to breakdown.) You
        now have enough glucose to start making more.
       PUT IN BOX – glycogen metabolism is regulated by the controlling activity of two key enzymes –
        the one that makes glycogen, glycogen synthase and the one that breaks it down, glycogen
        phosphorlyase.
       Regulated on many levels. One is allosteric and one is hormonally. Hormonally is mediated by
        reversible phosphorylation. Here is an example of that; mentioned by Miller.
       Idea is that phosphorlyase b is usually inactive. Once you phosphorlyate, it is usually active.

Slide 22
 This is hormonal regulation.
 A hormone can lead to increased phosphorylation.
 One of the hormones is GLUCAGON.
Slide 23
 The idea is phorphorylase B does NOT have a phosphate group.
 This can be in the relaxed for the tense state.
 Phorphorylase A can also be in the relaxed or tense state.
 The tense state is inactive in both cases.
 The relaxed state is active in both cases.
 The Key: Look at the arrows trying to illustrate that phosphorylase B, most of it, is in the tense
    state, i.e. the long arrow.
 Most of Phosphorylase A is in the active state, with the arrow being longer toward the active
    confirmation, the relaxed state.
Slide 24
 Phosphorylase kinase adds the phosphate.
 REMEMBER THIS ENZYME: PHOSPHORYLASE KINASE.
 This enzyme adds a phosphate to make phosphorylase B into phosphorylase A.
Slide 25
 Says he doesn’t remember the difference between this slide and previous one.
 Just go onto the next slide.
Slide 26
 The Key: it turns out in the MUSCLE and LIVER phosphorylase is regulated differently.
 In muscle, if you are running out of energy, what happens is that ATP is being converted to AMP.
 What do you want to go in the muscle? Get more ATP!
 To do this, you muscle breakdown the muscle glycogen.
 Normally, phosphorylase B is inactive; however, if there is a lot of AMP around (there will be a lot of
    AMP around if you have used up the ATP), what happens is that AMP binds and changes from the
    tense state to the relaxed state.
 The normally inactive phosphorylase B is now active.
 This can be reversed once ATP levels start to increase
 Main idea: Need more ATP turn the normally inactive phosphorylase B into the active one.
 Start breaking down glycogen to generate glucose to get more ATP.
Slide 27
 In the liver, this works differently.
 Liver glycogen has a different function.


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   When glucose levels rise, you don’t want to breakdown the liver glycogen because you don’t need
    any more glucose.
 The normally active phosphorylase A (which breaks down glycogen) should be turned down.
 When glucose binds, it causes phosphorylase A to enter the tense state and be inactivated.
 In the last two slides, we have been talking about allosteric regulation.
 The reversal of phosphorylation is hormonal regulation- it’s allosteric.
 Allosteric refers to the conformational change in an enzyme when it binds to a regulatory molecule.
Slide 28
 Skip
Slide 29
 Phosphorylase Kinase is the enzyme that phosphorylates phosphorylase.
 It is regulated too by being both phosphorylated and by binding Calcium.
 Phosphorylase kinase is a big and massive enzyme of 16 subunits- 4 of one type and 4 of another.
 Some of the subunits bind calcium and some can be phosphorylated.
 The enzyme can be partly activated by phosphorylation and partly active by binding calcium.
 When calcium is bound and it is also phosphorylated, you get an active enzyme that can now
    phosphorylate phosphorylase and convert the normally inactive phosphorylase b into phosphorylase
    a.
Slide 30
 This may seem like it is starting to get complicated, but it gets worse.
 PKA= protein kinase A  the enzyme that does the phosphorylation.
 Whether things are phosphorylated or dephosphorylated depends on the tight regulation of
    hormones.
Slide 31
 Epinephrine is the same as adrenaline.
 Tiny amounts of epinephrine in the blood have the effect of causing the activation of an enzyme,
    adenylyl cyclase, which makes cyclic AMP.
 Epinephrine is called the 1st messenger; it binds to receptors on the surface of the cell and triggers a
    series of steps that is called signal transduction.
 Signal transduction leads to the activation of an enzyme, adenylyl cyclase.
Slide 32
 When adenylyl cyclase adds to ATP, cyclic AMP is produced.
 Cyclic AMP is called the 2nd messenger.
 The hormone is the 1st messenger and comes through the blood and binds to the receptor.
 Cyclic AMP is made inside the cell and has effects on other enzymes.
 It can enormously active protein kinase A.
Side 33
 Protein kinase A is normally inactive.
 It is shown here as a tetramer.
 There are two catalytic subunits and 2 regulatory subunits.
 When lots of cyclic AMP is made, it binds to the regulatory subunits and they dissociate from the
    catalytic subunits and is now active.
 The regulatory subunit is an example of a modulator protein it has no enzyme activity itself but
    influences the activity of another subunit.
Slide 34
 Why do we have all these steps? Why protein kinase A, phosphorylase kinase, etc?


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   Why all these steps to regulate glycogen breakdown? One word answer amplification.
   If you are really excited about a sporting event or running from a bear, your epinephrine levels will
    go up when they are released in your blood.
 It will activate adenylyl cyclase which makes more cyclic AMP.
 When the enzyme is activated, it doesn’t just make one cyclic AMP, it operates to make hundreds
    and thousands of copies of cyclic AMPs.
 The cyclic AMP binds to active protein kinase A.
 An active protein kinase A will start phosphorylating phosphorylase kinase.
 One enzyme can phosphorylate hundreds and hundreds of phosphorylase kinase. At each step, you
    are getting tons of amplification.
 Get thousand and ten thousand fold amplification from just a tiny bit of hormone eventually
    actives phorphorylase A WHICH WILL BREAK DOWN LOADS AND LOAD OF GLYCOGEN.
 A lit bit of hormone in blood release a lot of glucose from the glycogen granules and get lots of
    energy for enormous bursts of energy if you need it.
Slide 35
 Similarly, this part in the box is the same sequence as the previous slide, but if you are going to be
    breaking glycogen down, the last thing you are going to want to be doing is wasting your time
    making more of it.
 This increase in phosphorylation activity has the effect of phosphorylating glycogen synthase.
 When glycogen synthase is phosphorylated, it is INACTIVATED.
 You don’t want to synthesize glycogen when you are busy breaking it down or you are just spinning
    your wheels.
Slide 36
 The net of effect of all the enzymes in the cascade (the previous two slides were activation
    cascades), is to cause increased phosphorylation.
 There is an important enzyme called Protein phosphatase 1 (PP1) which reverses all these things.
 It takes off the phosphates.
 If you are trying to breakdown glycogen fast, the last things you want is any of this enzyme around
    that is active.
 What happens is protein kinase A (which is responsible near the top of the cascade) does a couple
    things.
 It binds to a certain position on the regulatory subunit which links the PP1 to the glycogen and
    phosphorylates it and the PP1 falls off.
 It is now much, much less active.
 Basically trying to shut this down when you are in the process of breaking down glycogen because
    you don’t want this enzyme active because it will reverse all the effects that the phosphorylation
    results in.
 Similarly PKA can phosphorylate this inhibitor molecule. When this happens, it changes its
    confirmation so that it now binds to this PP1 and totally inactivates it.
 You have totally shut down this enzyme because this enzyme goes up when insulin levels increase.
 Insulin levels go up when you have just eaten a big meal and you have lots of glucose in your blood.
 The two hormones that have opposite effects are glucagon (glycogen breakdown) and insulin
    (glycogen synthesis).
 They have opposite effects.
Slide 37
 People and animals regulate their glucose levels carefully, between 80-120 mg/ml.


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   For historical reasons, blood chemistry values in medicine are always per 100 ml or deciliter  very
    weird units.
 If you are trying to regulate your blood glucose levels to an mg/ml, you need a way to sense what
    the glucose levels are.
 The glucose sensor is phosphorylase a (this breaks down glycogen).
 When glucose levels increase in the liver, glucose will then bind to phosphorylase a and switch it
    from the relaxed to tense from, which inactivates it.
 When glucose levels are high, you don’t want to break down glycogen in the liver to get more
    glucose because you already have enough.
 When glucose levels drop, then glucose no longer binds to this and you start breaking down
    glycogen again in order to maintain glucose levels.
 In addition, when glucose binds, it causes the phosphate group in phosphorylase to be exposed to
    PP1 which clips off the phosphate which further inactivates the phosphorylase a.
 PP1 can then begin to dephosphorylate glycogen synthase which actives it. When you
    dephosphorylate glycogen synthase, it comes active again. When it is phosphorylated, it is inactive.
Slide 38
 This is what happens to a rat when you infuse glucose into it and measure the activity of the
    enzymes afterwards.
 In a matter of minutes after glucose is added, the phosphorylase levels drop because you got
    glucose in the blood and glycogen does not need to be broken down.
 Glycogen synthase levels increase.
Slide 39
 When insulin levels go up after you eat a big meal and get lots of glucose in your blood, a bunch of
    steps occur.
 The net effect is that insulin sensitive protein kinase is activated.
 It phosphorylates the regulatory subunit at a different position than protein kinase A.
 The net affect here is an activation of the phosphatase.
 The phosphastase reverses all the effect of phosphorylation because it takes off all the phosphates.
Slide 40
 THIS IS AN IMPORTANT SLIDE: REMEMBER THESE THINGS BECAUSE THEY ARE TYPICAL BOARD EXAM
    QUESTIONS.
 Epinephrine and glucagon, while they lead to glycogen breakdown, they do it differently
 Epinephrine results in the breakdown of glycogen of BOTH MUSCLE AND LIVER.
 Glucagon has a different role than epinephrine.
 Epinephrine is the fight or flight hormone.
 Glucagon’s role is to maintain blood glucose levels.
 Glucagon DOES NOT have any effect of glycogen breakdown in muscle important to remember.
Slide 41
 This is showing how fast muscle glycogen can be broken down during exercise.
 Not much effect with light or moderate exercise.
 You can almost completely deplete your muscle glycogen in a couple of hours, maybe only one hour
    with very heavy exercise.
Slide 42
 How can you replenish your glycogen? It depends on what you eat.
 It you eat a high carb diet, in a matter of hours, you start replenishing your glycogen levels, even
    though it still takes a while to get way back up.


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   If you are on a low carb diet, it hardly goes up at all even after 5 days.
   What’s the take home message: If you are an athlete, don’t be on a low-carb diet because you need
    the glycogen for your energy.
Slide 43
 There is a disease with enzyme deficiencies.
 These enzyme deficiencies are important clues to biochemists about how things work.
 Some people are born without active glucose-6-phosphatase.
 Car and Gerti Cori figured this out (husband and wife biochemist team that figured out the Cori
    cycle).
 People with this disease have big, enlarged abdomen and thin extremities and abnormal blood lipids
    and abnormal blood sugar and all kinds of other problems.
 They don’t make an active glucose-6-phosphatase.
 This is the enzyme that is normally in the liver and kidneys that clip of the phosphate from glucose
    which lets the glucose get into the blood stream.
 These people can breakdown their glycogen but they can’t turn it into glucose. They break it down
    and can end up with G-1-P or G-6-P but can’t get the phosphate off.
 They have to use that in anaerobic glycolysis because the glucose can’t be put in the blood stream.
 They get enormous levels of lactic acid from this anaerobic glycolysis in the blood stream.
 The treatment for this condition is to give people small amount of glucose every few hours even if
    they have to be woken up at night every few hours.





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