Transcript PL05_Glucdisp

glucose
GLUTs
glucose
GLYCOGENESIS
G6P
GS – glycogen synthase
PFK – phosphofructokinase
GLYCOLYSIS
glucose
Muscle & WAT
Glucose Uptake
Translocation
insulin
Vesicles in Golgi
Hexose Metabolism
hexokinase
P
Using UTP
Releases PP
PP hydrolysis pulls
reaction to completion
P
glucose
Using ATP
glucose
6-phosphate
glucose
1-phosphate
PP
P
P
fructose
6-phosphate
P
PFK
fructose
1,6-bisphosphate
U
UDP glucose
“Activated Glucose”
Pyrophosphate hydrolyses to two phosphates
Pulls UDP-glucose conversion over
Glycogen Synthesis
PP
Glycogen
U
UDP glucose
PP
Glycogen with one more glucose
U
UDP
UDP needs to be made back into UTP
Use ATP for this
UDP + ATP  UTP + ADP
Note synthesis is C1 C4
C1 end of glycogen attached to glycogenin
Glycogen Synthase
• Catalyses the addition of ‘activated’ glucose onto an
existing glycogen molecule
– UDP-glucose + glycogenn UDP + glycogenn+1
• Regulated by reversible phosphorylation (covalent
modification)
– Active when dephosphorylated, inactive when phosphorylated
• Phosphorylation happens on a serine residue
– Dephosphorylation catalysed by phosphatases (specifically
protein phosphatase I)
– Phosphorylation catalysed by kinases (specifically glycogen
synthase kinase)
• Insulin stimulates PPI
– And so causes GS to be dephosphorylated and active
– So insulin effectively stimulates GS
Phosphofructokinase
• Catalyses the second ‘energy investment’ stage
of glycolysis
– F6P + ATP  fructose 1,6 bisphosphate + ADP
• Regulated allosterically
– Simulated by concentration changes that reflect a low
energy charge
• An increase in ADP/AMP and a decrease in ATP
• These molecules bind at a site away from the active site –
the allosteric binding sites.
– Many other molecules affect PFK allosterically but all
are effectively indicators of ‘energy charge’
• Energy charge is balance of ATP, ADP & AMP
• Small change in ATP/ADP causes large change in AMP via
adenylate kinase reaction
Coupling (again!)
• The stimulation of glycogen synthesis by insulin creates
an ‘energy demand’
– Glycogenesis is anabolic
– The activation of glucose prior to incorporation into glycogen
requires ATP
– This drops the cellular [ATP] and increases the [ADP] & [AMP]
• This drop in ‘energy charge’ is reflected by a stimulation
of PFK
– A good example of how an anabolic pathway requires energy
from a catabolic pathway
– Insulin has ‘indirectly’ stimulated PFK and glucose oxidation
even though it does not have any direct lines of communication
to this enzyme
– Signals to store fuels also cause fuels to be burnt
Liver Glucose Uptake
• GLUT-2 used to take up glucose from bloodstream
– Very high activity and very abundant
– [Glucose] blood = [Glucose] liver
• Glucokinase
– Rapidly converts GG6P
– Not inhibited by build up of G6P
– High Km (10 mM) for glucose – not saturated by high levels of
liver glucose
– So [G6P] rapidly increases as blood [glucose] rises
• G6P can stimulate inactive GS
– Even phosphorylated GS
– Glucose itself also stimulates the dephosphorylation of GS
• Via a slightly complex process that involves other kinases and
phosphatases which we needn’t go into right now 
Glycogenesis
• In liver
– The “push” mechanism
• Glycogenesis responds to blood glucose without the need of insulin
• Although insulin WILL stimulate glycogenesis further
• In muscle
– [G6P] never gets high enough to stimulate GS
• “Push” method doesn’t happen in muscle
• More of a “pull’ as insulin stimulates GS
• In both cases
– 2 ATPs required for the incorporation of a glucose into glycogen chain
• GG6P and UDPUTP
– Branching enzyme needed to introduce a16 branch points
– Transfers a segment from one chain to another
– Limit to the size of glycogen molecule
• Branches become too crowded, even if they become progressively shorter
• Glycogen synthase may need to interact with glycogenin to be fully active
A Tale of Two Kinases
• Glucokinase (GK)
–
–
–
–
–
Only works on glucose
High Km for glucose (~10mM)
Not inhibited by G6P
Only presents in liver, beta-cells
Responsive to changes in [glucose] blood
• Hexokinase (HK)
–
–
–
–
–
Works on any 6C sugar
Km for glucose ~0.1mM
Strongly inhibited by its product G6P
Present in all other tissues
If G6P is not used immediately, its build up and inhibits
hexokinase
– Easily saturated with glucose
Fructose Metabolism
Glyceraldehyde
fructokinase
fructose
Using ATP
hexokinase
P
fructose
1-phosphate
Glyceraldehyde 3phosphate
Aldolase CH2OH
B
CHOH
CHO
CH2OP
CHOH
Triose
Kinase
CHO
CH2OP
P
C=O
CH2OH
fructose
6-phosphate
Dihydroxyactone
phosphate
PFK
‘normal glycolysis’
‘normal glycolysis’
Fructose Metabolism
• Fructose entry into cells does not require insulin
• In muscle, fructose just enters glycolysis
– Or could be made into glycogen if insulin stimulus available!
• F6P  G6P  G1P  UDP-glucose  Glycogen
• In liver, fructokinase traps fructose
– FK produces F1P
– FK is quite fast in comparison to the aldolase B that uses the
F1P
– F1P can build up
– But more seriously producing ‘dead’ F1P traps phoshpate
• FK reaction consumes ATP
• Lack of phosphate akes new ATP synthesis difficult
• ATP levels in liver fall
– Even more serious in people with a deficiency in Aldolase B
Lipogenesis Overview
glucose
Fat
ESTERIFICATION
GLUT-4
No GS
glucose
X
fatty acids
G6P
PPP
Consumes
reductant
and ATP
GLYCOLYSIS
Produces
reductant
LIPOGENESIS
pyruvate
acetyl-CoA
pyruvate
acetyl-CoA
PDH
Key steps (eg, GLUT-4, PDH,
lipogenesis) are stimulated when
insulin binds to its receptor on
the cell surface
KREBS
CYCLE
CO2
NADH release
ultimately
produces ATP
Pyruvate Dehydrogenase
Pyruvate + CoA + NAD  acetyl-CoA +
NADH + CO2
• Irreversible in vivo
• No pathways in humans to make acetate
into ‘gluconeogenic’ precursors
– Can’t make glucose from acetyl-CoA
– No way of going back once the PDH reaction
has happened
– Key watershed between carbohydrate and fat
metabolism
PDH Control
• Regulated by reversible phosphorylation
– Active when dephosphorylated
• Inactivated by PDH kinase
• Activated by PDH phosphatase
– Insulin stimulates PDH phosphatase
• Insulin thus stimulates dephosphorylation and
activation of PDH
Fate of Acetyl-CoA
• Burnt in the Krebs Cycle
– Carbon atoms fully oxidised to CO2
– Lots of NADH produced to generate ATP
• Lipogenesis
– Moved out into the cytoplasm
– Activated for fat synthesis
• In both cases the first step is citrate formation
– Condensation of acetyl-CoA with oxaloacetate
• Regenerates Coenzyme A
– Transport or Oxidation
• The ‘fate’ will depend on the need for energy (ATP/energy
charge) and the stimulus driving lipogenesis
ATP-Citrate Lyase
• Once in the cytoplasm, the citrate is cleaved
– By ATP-Citrate Lyase (ACL)
– Using CoA to generate acetyl-CoA and oxaloacetate
• Reaction requires ATP  ADP + phosphate
• ACL is inhibited by hydroxy-citrate (OHCit)
– OHCit is found in the Brindleberry
• Sold as a fat synthesis inhibitor
– Would we expect it to prevent the formation of fatty
acids
• And, if so, would that actually help us lose weight?
The Carrier
• Oxaloacetate produced by ACL needs to return
to the matrix
– Otherwise the mitochondrial oxaloacetate pool
becomes depleted
– Remember, oxaloacetate is really just a ‘carrier’ of
acetates
• Both in the Krebs's cycle and in the transport of acetyl-CoAs
into the cytoplasm
– Oxaloacetate cannot cross the inner mitochondrial
membrane
• Some interesting inter-conversions occur to get it back in!
Acetyl-CoA Carboxylase
• Activates acetyl-CoA and ‘primes’ it for
lipogenesis
• Unusual in that it ‘fixes’ carbon dioxide
– In the form of bicarbonate
– A carboxylation reaction
Acetyl-CoA + CO2  malonyl-CoA
– Reaction requires ATP  ADP + phosphate
– Participation of the cofactor, biotin
• Biotin is involved in other carboxylation reactions
ACC Control
• ACC is stimulated by insulin
– Malonyl-CoA is committed to lipogenesis
• Reversible Phosphorlyation
• Stimulated allosterically by citrate
(polymerisation)
• Inhibited allosterically by long-chain fatty
acyl-CoAs
Malonyl-CoA
• Activated acetyl-CoA
–
–
–
–
Tagged and primed for lipogenesis
But also a key regulator of fatty acid oxidation
ACC is not only present in lipogenic tissues
Also present in tissues that need to produce malonylCoA in ‘regulatory’ amounts
• Malonyl-CoA inhibits carnitine acyl transferase I
– An essential step in fatty acid oxidation
– Only way of getting long chain fatty acyl-CoAs into the
mitochondria
Malonyl-CoA
• So when ACC is active in, say, muscle
– Malonyl-CoA concentration rises
– CPT-1 is inhibited
– Fatty acid oxidation stops
– Cell must use carbohydrate instead
– Therefore insulin, by stimulating acetyl-CoA
carboxylase, encourages carbohydrate
oxidation and inhibits fatty acid oxidation
Fatty Acyl Synthase
FAS - simplified
FAS
• Fatty acyl synthase (FAS) is multi-functional
– Lots of different enzyme activities in the complex
– Can you count them all?
• Bringing in acetyl and malonyl groups, catalysing the reaction
between the decarboxylated malonyl and the growing fatty acid
chain, the reduction/dehydration/reduction steps, moving the
fatty acid to the right site and finally releasing it as FA-CoA
• Two free -SH groups on an ‘acyl-carring protein’
– Keeps the intermediates in exactly the right position for
interaction with the right active sites
– Each new 2C unit is added onto the carboxy-end
Addition Sequence
• Each round of 2C addition requires
– 2 molecules of NADPH … but No ATP (!!)
– The release of the carbon dioxide that went on
during the production of malonyl-CoA
• Thus the carboxylation of acetyl-CoA does not result
in ‘fixing’ CO2
• FAs start getting ‘released’ as FA-CoA
when chain length is C14
– Desaturation is done AFTER FAS
Pentose Phosphate Pathway
• Provides NADPH for lipogenesis
– NADPH - A form of NADH involved in anabolic
reactions
– Rate of NADPH production by PPP is proportional to
demand for NADPH
• Key regulatory enzyme is G6PDH
– Glucose 6-phosphate dehydrogenase
G6P + NADP  6-phosphogluconolactone + NADPH
– The gluconolactone is further oxidised to give more
NADPH
• Decarboxylation to give a 5-carbon sugar phosphate (ribulose
5-phosphate)
Pentose Phosphate Pathway
• Need to put the 5-C sugar back into glycolysis
– Accomplished by rearranging and exchanging carbon atoms
between 5C molecules
– Catalysed by enzymes called transaldolases and transketolases
• So, 5C + 5C  C7 + C3 by a transketolase (2C unit transferred)
• Then C7 + C3  C6 + C4 by a transaldolase (3C unit transferred)
• Then C4 + C5  C6 + C3 by a transketolase (2C unit transferred)
– The C6 and C3 sugars can go back into glycolysis
• Alternatively, PPP used to make ribose 5-phosphate
– Important in nucleotide pathways
• Or generate NADPH as an anti-oxidant
– Red blood cells - deficiency in G6PDH can cause anemia
Esterification
• Formation of Fat
• Glycerol needs to be glycerol 3-phosphate
– From reduction of glycolytic glyceraldehyde 3-phosphate
– Glycolysis important both for production of acetyl-CoA and
glycerol!
• Esterification enzyme uses FA-CoA
– Not just FAs
– FAs added one at a time
• Both esterification enzyme and FAS are unregulated by
insulin
– Gene expression and protein synthesis
• FAS is downregulated when lots of fat around
– As in a Western diet!!
Regulatory Overview
ESTERIFICATION
glucose
GLUT-4
glucose
No GS
X
fatty acids
G6P
G6PDH
Fat
glycerol 3-P
FAS
LIPOGENESIS
GLYCOLYSIS
ACC
pyruvate
acetyl-CoA
pyruvate
acetyl-CoA
PDH
G6PDH stimulated by demand for NADP
Insulin stimulates GLUT-4. PDH and
ACC. Also switches on the genes for
FAS and esterification enzyme.
Krebs cycle will be stimulated by demand for ATP
citrate
KREBS
CYCLE
CO2
Acetyl-CoA
transport
stimulated by
increased
production of
citrate