Carbohydrate Disposal

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Carbohydrate Disposal

• This version is quite information dense to save
  paper.

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Sources of Dietary Carbs

• Starch polymer of glucose
• Amylose
• linear, forms helices, difficult to digest,
  flatulence
• Amylopectin
• branched, easy to digest

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Sources of Dietary Carbs

• Disaccharides
• Lactose
• galactose and glucose
• consequences of lactase deficiency lactose
  intolerance
• Sucrose
• fructose and glucose
• Maltose
• glucose and glucose
• Monosaccharides
• Glucose
• Fructose
• especially these days with high fructose corn
  syrup

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Glucose responses
Results of consuming a standard 50 g glucose load
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Intolerant
Blood Glucose (mM)
Tolerant
5
0
1
2
Time (h)
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Consequences of Intolerance

• Post-prandial hyperglycemia is a problem
• If occurs after each meal and persists for
  several hours then there will be problems
• The person will rarely be euglycemic!
• Leads to complications of hyperglycemia
• Protein glycosylation
• Root cause may be insulin resistance
• Impaired ability of tissues to respond to insulin
• Underlies Type II Diabetes
• Control of glucose intolerance
• Consumption of slowly absorbed starches

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Starch Digestion
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Different Glycemic Responses
Amylopectin
Blood Glucose (mM)
Amylose
5
0
1
2
Time (h)
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The Glycemic Index

• Describes the post-prandial glucose response
• Area under the test food glucose curve divided
  by
• Area under a reference food glucose curve
• Reference food is normally 50 g gluocse
• Test food given in an amount that will give 50 g
  digestible carbohydrate
• Expressed as a
• GI of modern, processed, amylopectin foods gt80
• GI of legumes lt 30

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The Glycemic Index

• Useful knowledge for controlling blood glucose
• Especial relevance to diabetes
• QUALITY of carbohydrate (GI) as important as
  total amount of carbohydrate

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GI critics say..

• Area under slowly absorbed may be the same as
  quickly absorbed
• Look closely at previous figure
• The GI should not apply to foods other than
  starches
• Sugary foods are low GI
• Because half the carbohydrate is fructose
• Similarly, fructose containing foods are low GI
• Dairy foods are low GI
• Because half the carbohydrate is galactose
• And protein elicits insulin secretion ?
  lipogenesis

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GI critics say..

• Some Low GI values
• Due to inaccurate estimation of digestible
  carbohydrate portion
• Claims of slow burning energy ??
• What regulates energy expenditure and supply of
  substrates?
• Even if supply was important, the classic
  persistently but subtly raised post-prandial
  glucose response is hardly ever seen

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Muscle WAT Glucose Uptake
glucose
GLUTs
GLYCOGENESIS
GS glycogen synthase
glucose
G6P
PFK phosphofructokinase
GLYCOLYSIS
glucose
Translocation
Vesicles in Golgi
insulin
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Hexose Metabolism
P
hexokinase
Using UTP Releases PP PP hydrolysis pulls
reaction to completion
P
Using ATP
glucose
glucose 1-phosphate
glucose 6-phosphate
P
P
P
U
UDP glucose
fructose 6-phosphate
P
P
Activated Glucose
PFK
Pyrophosphate hydrolyses to two phosphates Pulls
UDP-glucose conversion over
fructose 1,6-bisphosphate
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Glycogen Synthesis
P
P
Glycogen
U
UDP glucose
P
P
Glycogen with one more glucose
U
Note synthesis is C1? C4 C1 end of glycogen
attached to glycogenin
UDP
UDP needs to be made back into UTP Use ATP for
this UDP ATP ? UTP ADP
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Glycogen Synthase

• Catalyses the addition of activated glucose
  onto an existing glycogen molecule
• UDP-glucose glycogenn? UDP glycogenn1
• 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, PPI)
• 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

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Phosphofructokinase

• Catalyses the second energy investment stage of
  glycolysis
• F6P ATP ? fructose 1,6 bisphosphate ADP
• Regulated allosterically
• Simulated by low energy charge
• Energy charge is balance of ATP, ADP AMP
• 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.
• Small change in ATP/ADP causes large change in
  AMP via adenylate kinase reaction
• Many other molecules affect PFK allosterically
  but all are effectively indicators of energy
  charge

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Coupling (again!)

• The stimulation of glycogen synthesis by insulin
  creates an energy demand
• Glycogenesis is anabolic
• The activation of glucose requires ATP
• This drops the cellular ATP and increases the
  ADP AMP
• Drop in energy charge is stimulates PFK
• Anabolic pathway requires catabolic pathway
• Insulin has indirectly stimulated PFK and
  glucose oxidation
• So signals to store fuels also cause fuels to be
  burnt

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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 neednt
  go into right now ?

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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 doesnt happen in muscle
• More of a pull as insulin stimulates GS

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Glycogenesis

• In both liver and muscle
• 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

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Hexokinases

• 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

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Lipogenesis Overview
glucose
Fat
ESTERIFICATION
GLUT-4
No GS
X
fatty acids
glucose
G6P
Consumes reductant and ATP
GLYCOLYSIS
PPP
LIPOGENESIS
Produces reductant
pyruvate
acetyl-CoA
acetyl-CoA
pyruvate
PDH
Key steps (eg, GLUT-4, PDH, lipogenesis) are
stimulated when insulin binds to its receptor on
the cell surface
KREBS CYCLE
NADH release ultimately produces ATP
CO2
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Pyruvate Dehydrogenase

• Pyruvate CoA NAD ? acetyl-CoA NADH CO2
• Irreversible in vivo
• No pathways in humans to make acetate into
  gluconeogenic precursors
• Cant make glucose from acetyl-CoA
• No way of going back once the PDH reaction has
  happened
• Key watershed between carbohydrate and fat
  metabolism

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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

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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

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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?

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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!

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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

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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

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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
  malonyl-CoA 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

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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

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Fatty Acyl Synthase
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FAS - simplified
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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

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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

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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)

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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

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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!!

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Regulatory Overview
Fat
glucose
ESTERIFICATION
GLUT-4
No GS
X
fatty acids
glucose
G6P
G6PDH
glycerol 3-P
FAS
LIPOGENESIS
GLYCOLYSIS
ACC
pyruvate
acetyl-CoA
Acetyl-CoA transport stimulated by increased
production of citrate
acetyl-CoA
pyruvate
PDH
citrate
G6PDH stimulated by demand for NADP
KREBS CYCLE
Insulin stimulates GLUT-4. PDH and ACC. Also
switches on the genes for FAS and esterification
enzyme.
CO2
Krebs cycle will be stimulated by demand for ATP