Glycogen Metabolism and Gluconeogenesis

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Glycogen Metabolism andGluconeogenesis

• CH 339K

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Glycolysis (recap)

• We discussed the reactions which convert glucose
  to pyruvate
• C6H12O6 2 NAD 2 ADP ? 2 CH3COCOOH 2 NADH 2
  ATP 2 H
• What about the sources of glucose?
• Dietary sugars
• Glycogen

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Before we get to glycogen Dietary sugars
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Amylase Reaction
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Glycogen

• Branched every 8-12 residues
• Up to 50,000 or so residues total

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Breakdown Glycogen Phosphorylase
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Glycogen Synthesis and Breakdown

• Glycogen synthesis and breakdown are both
  controlled by hormones
• Glucagon, Epinephrine
• turn on glycogen breakdown
• Turn off glycogen synthesis
• Hormones act through receptors on cell surface
  and G-proteins

Glucagon 29 amino acid polypeptide produced in
pancreas in response to low blood sugar
Epinephrine aka adrenaline produced by
adrenal medulla in response to stress
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Activation of Glycogen Phosphorylase

• G-Proteins
• Second messengers
• Kinase Cascade

3-5 cyclic AMP
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G-Proteins

• G proteins are heterotrimers, containing Ga, Gb
  and Gg subunits.

Subunit Size
Ga 45 47 kD
Gb 35 kD
Gg 7-9 kD
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G-Proteins

• The Ga subunits bind guanine nucleotides (hence
  the name G Protein). G Proteins are associated
  on one hand with the inner surface of the plasma
  membrane, and on the other hand with membrane
  spanning receptor proteins called G-protein
  coupled receptors or GPCRs.
• There are a number of different GPCRs most
  commonly these are receptors for hormones or for
  some type of extracellular signal.
• In the resting state, Ga is bound to the Gb-Gg
  dimer. Ga contains the nucleotide binding site,
  holding GDP in the inactive form, and is the
  warhead of the G protein. At least 20
  different forms of Ga exist in mammalian cells.
• Binding of the extracellular signal by the GPCR
  causes it to undergo an intracellular
  conformational change this causes an allosteric
  effect on Ga. The change in Ga causes it to
  exchange GDP for GTP. GTP activates Ga, causing
  it to dissociate from the Gb-Gg dimer. The
  activated Ga binds and activates an effector
  molecule.
• Ga also has a slow GTPase activity. Hydrolysis
  of GTP deactivates Ga, which reassociates with
  the Gb-Gg dimer and the GPCR to reform the
  resting state. In other words, G-protein
  mediated cellular responses have a built-in off
  switch to prevent them from running forever.

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G-Protein Coupled Receptors (GPCRs)
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G-Proteins Effect of GDP/GTP Binding
GTP terminal PO4 constrains the bg-binding loop
(red)
GDP missing terminal PO4 allows the bg-binding
loop (red) to assime a looser conformation
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Cycling of G protein between active and inactive
states
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G-Protein Killers

• Cholera
• Cholera toxin secreted by the bacterium Vibrio
  cholera.
• A subunit and five B subunits.
• A subunit catalyzes the transfer of an ADP-ribose
  from NAD to a specific Arg side chain of the a
  subunit of Gs.
• Ga is irreversibly modified by addition of
  ADP-ribosyl group
• Modified Ga can bind GTP but cannot hydrolyze it
  ).
• As a result, there is an excessive, nonregulated
  rise in the intracellular cAMP level (100 fold or
  more), which causes a large efflux of Na and
  water into the gut.
• Pertussis (whooping cough)
• Pertussis toxin (secreted by Bordetella
  pertussis) catalyzes ADP-ribosylation of a
  specific cysteine side chain on the a subunit of
  a G protein which inhibits adenyl cyclase and
  activates sodium channels.
• This covalent modification prevents the subunit
  from interacting with receptors as a result,
  locking Ga in the GDP bound form.
• You probably vaccinate your dog against the
  related species that causes kennel cough.

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Cholera is still a problem-2009 Zimbabwe
outbreak 4300 deaths
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Activation of Adnylate Cyclase
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Activation of cAMP-Dependant Protein Kinase
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Glycogen Phosphorylase

• Exists in 2 forms
• Phosphorylase B (inactive)
• Phosphorylase A (active)
• Phosphorylase B is converted to Phosphorylase A
  when it is itself phosphorylated by Synthase
  Phosphorylase Kinase (SPK)
• GP cannot remove branch points (a-1,6 linkages)

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Activation of Glycogen Phosphorylase
cAMP dependent Protein Kinase
3-5 cyclic AMP
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Activation of Glycogen Phosphorylase
PLP Pyridoxal Phosphate cofactor
cAMP dependent Protein Kinase
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Debranching Enzyme

• The activity of phosphorylase ceases 4 glucose
  residues from the branch point.
• Debranching enzyme (also called glucan
  transferase) contains 2 activities
• glucotransferase
• glucosidase.
• Glycogenolysis occurring in skeletal muscle could
  generate free glucose which could enter the blood
  stream.
• However, the activity of hexokinase in muscle is
  so high that any free glucose is immediately
  phosphorylated and enters the glycolytic pathway.
  

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Cori Disease

• Cori disease (Glycogen storage disease Type III)
  is characterized by accumulation of glycogen with
  very short outer branches, caused by a flaw in
  debranching enzyme.

• Deficiency in glycogen debranching activity
  causes hepatomegaly, ketotic hypoglycemia,
  hyperlipidemia, variable skeletal myopathy,
  cardiomyopathy and results in short stature.

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Glycogen Synthesis

• Glycogen Synthase adds glucose residues to
  glycogen
• Synthase cannot start from scratch needs a
  primer
• Glycogenin starts a new glycogen chain, bound to
  itself

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Glycogen Synthesis (cont.)

• Synthase then adds to the nonreducing end.

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Glycogen Synthesis (cont.)

• To add to the glycogen chain, synthase uses an
  activated glucose, UDP-Glucose
• UDP-Glucose Pyrophosphorylase links UDP to glucose

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Glycogen Synthesis (cont.)

• Synthase then adds the activated glucose to the
  growing chain
• Release and subsequent hydrolysis of
  pyrophosphate drives the reaction to the right

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Glycogen Synthesis (cont.)

• Glycogen branching enzyme then introduces branch
  points

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Mature Glycogen

• Built around glycogenin core
• Multiple non-reducing ends accessible to glycogen
  phosphorylase

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Reverse Regulation of Phosphorylase and Synthase

• The same kinase phosphorylates both glycogen
  phosphorylase and synthase
• Synthase I (dephos.) is always active
• Synthase D (phos.) is dependent on G-6-P
• The same event that turns one on turns the other
  one off.

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Gluconeogenesis

• CH 339K

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Gluconeogenesis

• Average adult human uses 120 g/day of glucose,
  mostly in the brain (75)
• About 20g glucose in body fluids
• About 190 g stored as glycogen
• Less than 2 days worth
• In addition to eating glucose, we also make it
• Mainly occurs in liver (90) and kidneys (10)
• Not the reverse of glycolysis
• Differs at the irreversible steps in glycolysis

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Gluconeogenesis
Differs Here
And Here
And Here
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First Difference
Gluconeogenesis burn two nucleotide triphosphates
Glycolysis make a nucleotide triphosphate
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Pyruvate Carboxylase
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PEP Carboxykinase
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Malate Shuttle

• Pyruvate Carboxylase is mitochondrial
• OAA reduced to malate in matrix
• Carrier transports malate to cytoplasm
• Cytoplasmic malate dehydrogenase reoxidizes to
  OAA
• Mammals have a mitochondrial PEPCK

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Second and Third differences
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Energetics

• Gluconeogenesis
• Pyruvate 4 ATP 2 GTP 2 NADH 2 H2O ?
  glucose 4 ADP 2 GDP 2 NAD
• ?G -37 kJ/mol
• Glycolysis (reversed)
• Pyruvate 2 ATP 2 NADH 2 H2O ? glucose 2
  ADP 2 NAD
• ?G 84 kJ/mol
• Net difference of 4 nucleotide triphosphate bonds
  at 31 kJ each accounts for difference in DGs
•  

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Local Regulation

• Phosphofructokinase-1(Glycolysis) is inhibited by
  ATP and Citrate and stimulated by AMP.
• Fructose-1,6-bisphosphatase (Gluconeogenesis) is
  inhibited by AMP.

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Global Control

• Enzymes relevant to these pathways that are
  phosphorylated by cAMP-Dependent Protein Kinase
  include
• Pyruvate Kinase, a glycolysis enzyme that is
  inhibited when phosphorylated.
• A bi-functional enzyme that makes and degrades an
  allosteric regulator, fructose-2,6-bisphosphate.

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Pyruvate Kinase Regulation

• Local regulation by substrate activation
• Global regulation by hormonal control of
  Protein Kinase A

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Effects of Fructose-2,6-Bisphosphate

• Fructose-2,6-bisphosphate allosterically
  activates the glycolysis enzyme
  Phosphofructokinase-1, promoting the relaxed
  state, even at relatively high ATP. Activity in
  the presence of fructose-2,6-bisphosphate is
  similar to that observed when ATP is low. Thus
  control by fructose-2,6-bisphosphate, whose
  concentration fluctuates in response to external
  hormonal signals, supercedes control by local
  conditions (ATP concentration).
• Fructose-2,6-bisphosphate instead inhibits the
  gluconeogenesis enzyme Fructose-1,6-bisphosphatase
  .

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Source of Fructose-2,6-Bisphosphate

• Fructose-2,6-bisphosphate is synthesized and
  degraded by a bi-functional enzyme that includes
  two catalytic domains
• Phosphofructokinase-2 (PFK2) domain
  catalyzesfructose-6-phosphate ATP ?
  fructose-2,6-bisphosphate ADP.
• Fructose-Biosphosphatase-2 (FBPase2) domain
  catalyzesfructose-2,6-bisphosphate H2O ?
  fructose-6-phosphate Pi.

Phosphorylation activates FBPase2 and inhibits
PFK2
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BifunctionalEnzyme
Activates PFK1 Inhibits F-1,6-bisphosphatase
Inhibits PFK1 Activates F-1,6-bisphosphatase
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Reciprocal Regulation of PFK-1 and FBPase-1
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Medical aside nonlethal!
People with Type II diabetes have very high (3x
normal) rates of gluconeogenesis Initial
treatment is usually with metformin.
Metformin shuts down production of PEPCK and
Glucose-6-phosphatase, inhibiting gluconeogenesis.
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Futile Cycles

• Occur when loss of reciprocal regulation fails
  twixt glycolysis and gluconeogenesis
• Anesthestics like halothane occasionally lead to
  runaway cycle between PFK and fructose-1,6-BPase
• Malignant Hyperthermia

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The Cori Cycle
High NADH/NAD
Low NADH/NAD