GLUCONEOGENESIS

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-- GLUCONEOGENESIS --   Definition the
biosynthesis of glucose from simpler molecules,
primarily pyruvate and its precursors.
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The gluconeogenesis pathway is similar to the
reverse of glycolysis but differs at critical
sites.  ?control of these opposing pathways is
reciprocal so that physiological conditions
favoring one disfavor the other and vice
versa.   ? General principles of
metabolic control -- a) pathways are not
simple reversals of each other and b) under
reciprocal control
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 Why do we produce glucose? a) Need to maintain
glucose levels in a narrow range in blood. b)
Some tissue-- brain, erythrocytes, and muscles in
exertion use glucose at a rapid rate and
sometimes require glucose in addition to dietary
glucose. c) The brain uses mostly glucose and
erythrocytes can use only glucose as a source of
energy.
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Where is glucose synthesized? The liver comes to
rescue. The liver is the major location for
gluconeogenesis.
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What are the sources of precursors? ? pyruvate-
major precursor ? lactatefrom muscle, forms
pyruvate ? some amino acid carbon skeletons- from
diet or breakdown of muscle protein during
starvation- most important is alanine ? TCA cycle
intermediates ? propionate from breakdown of
fatty acids and amino acids. ? glycerol from
certain lipids.
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Cost The production of glucose is energy
expensive. Input 2 pyruvate 4 ATP 2 GTP
2 NADH Output glucose 4 ADP 2 GDP
2 NAD 6 Pi
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Gluconeogenesis - beginning GDP CO2
phosphoenolpyruvate GTP
phosphoenolpyruvate
carboxykinase BYPASS 1 oxaloacetate
Some amino acids ADP Pi pyruvate
carboxylase ATP CO2
(?pyruvate kinase) PYRUVATE (3C) ?lactate,
alanine, other
amino acids
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fructose 1,6-bisphosphate
aldolase triose
phosphate isomerase dihydroxyacetone
glyceraldehyde phosphate
3-phosphate glyceraldehyde
NAD Pi 3-phosphate
NADH H dehydrogenase
1,3-bisphosphoglycerate

ADP phosphoglycerate kinase
ATP
3-phosphoglycerate phosphoglyceromutase
2-phosphoglycerate enol
ase
phosphoenolpyruvate
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GLUCOSE
glucose Pi BYPASS 3
6-phosphatase (?hexokinase)
Glucose 6-phosphate phosphogluco-
isomerase Fructose
6-phosphate BYPASS 2 fructose Pi
(?phospho- 1,6 bisphosphatase
fructokinase) Fructose 1,6-bisphosphate
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Pyruvate ? major precursor
for gluconeogenesis.
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? Lactate is the primary source for pyruvate. --
In muscle, lactate is produced in great
quantities during exertion. -- This excess
lactate cannot be further oxidized in
muscle.   -- Lactate is released from the muscles
to the blood and travels to the liver for
conversion to pyruvate and, ultimately to
glucose.
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In glycolysis, there are three irreversible
kinase reactions at control points
involving hexokinase, phosphofructokinase, and
pyruvate kinase
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?In gluconeogenesis, these reactions must be
forced the other way. ? The control points are
the same for gluconeogenesis and for
glycolysis. ? Four unique enzymes are used
to bypass these irreversible steps. ? The rest
of the steps use the same enzymes as glycolysis.
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Bypass number 1. Pyruvate to phosphoenolpyruvate.
This bypasses pyruvate kinase.   ? Complex
scheme.
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a) pyruvate to oxaloacetate Enzyme pyruvate
carboxylase   ? located inside mitochondria.
Only this enzyme of the gluconeogenesis pathway
is mitochondrial.
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? Reaction   pyruvate CO2 ATP H2O ?
oxaloacetate ADP Pi   -- Carboxylations
involving CO2 almost always use the vitamin
biotin as a cofactor.
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-- Subreactions   ? Enz-biotin ATP CO2 H2O
? Enz-carboxybiotin ADP Pi ?
Enz-carboxybiotin pyruvate ?
Enz-biotin oxaloacetate
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-- pyruvate carboxylase -- ? acetyl-CoA is
positive modulator ? absolutely required for
activity ? higher acetyl-CoA indicates
that adequate carbon levels available for TCA
cycle to provide energy ? glucose can be
synthesized and exported from liver. ?
oxaloacetate important in the citric acid cycle,
which is more mitochondrial.
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? For gluconeogenesis, oxaloacetate must leave
the mitochondria because all the rest of the
gluconeogenesis enzymes are in the cytosol.   ?
mitochondrial membranes are nearly impermeable to
oxaloacetate. So how does it get out?
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b) transport - three steps ? malate
dehydrogenase
mitochondrial enzyme   Oxaloacetate NADH H ?
malate (mitochondria)
NAD ? malate can be malate
oxaloacetate transported through HO-
O the mitochondrial C-COO-
membrane HCH
COO-
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? malate dehydrogenase -- cytosolic
enzyme malate
NAD ? oxaloacetate (cytosolic)
NADH H
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c) The GTP-dependent decarboxylation of
oxaloacetate Enzyme PEP carboxykinase
cytosolic enzyme,
as all others
oxaloacetate GTP?phosphoenolpyruvate
CO2 GDP ?
uses GTP, not ATP. ? CO2 added is lost in this
step. NET so far pyruvate ATP GTP ?
PEP ADP GDP Pi
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High cost two energy rich phosphates ? so a
total of four high energy bonds are already
utilized here per glucose to be synthesized.   ?
Then uses glycolytic enzymes in steps to
fructose-1,6-bisphosphate

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Bypass number 2. Fructose-1,6- bisphosphate to
fructose-6-phosphate Enzyme fructose-1,6-bisphos
phatase Reaction fructose-1,6-bisphosphate H2O
? fructose-6-P Pi ?
bypasses phosphofructokinase ? a simple
hydrolysis. ? highly exergonic, irreversible ?
enzyme is highly regulated F6P isomerizes to
glucose-6-phosphate via phosphoglucoisomerase
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Bypass number 3. Glucose-6-phosphate
to glucose.  
Enzyme glucose-6-phosphatase   Reaction
glucose-6-phosphate H2O ? glucose
Pi ? bypasses hexokinase ? highly exergonic,
irreversible ? not present in muscle
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Total
Energy Cost 6 high energy bonds used per
glucose synthesized. four more than produced in
glycolysis. These four are needed to
convert pyruvate to PEP.

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CONTROL   -- gluconeogenesis serves as
an alternative source of glucose when supplies
are low and is largely controlled by diet. --
high carbohydrate in meal reduce gluconeogenesis
and fasting increases. -- key enzymes
targeted. -- gluconeogenesis and glycolysis are
controlled in reciprocal fashion.
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REGULATION OF GLUCONEOGENESIS Key enzymes 1.
PYRUVATE CARBOXYLASE. ? activated by acetyl-CoA
(required) vs. pyruvate kinase, inhibited
by acetyl CoA. ? high levels of acetyl-CoA
signals that enough carbon substrate
available for citric acid cycle. ? pyruvate
kinase is also inhibited by ATP and the liver
form by alanine.
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2. FRUCTOSE 1,6 BISPHOSPHATASE   ? Strongly
inhibited by AMP, low energy ? Strongly inhibited
by fructose-2,6-bisphosphate,
high glucose ?  ?
Recall that the reciprocal enzyme, phosphofructoki
nase, in glycolysis, is strongly activated by AMP
and fructose-2,6-bisphosphate.
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Fructose-2,6-bisphosphate is the most important
regulator of glycolysis and gluconeogenesis
through its reciprocal effects on fructose
1,6-bisphosphatase and phosphofructokinase.
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3. PHOSPHOFRUCTOKINASE-2/ FRUCTOSE
BISPHOSPHATASE-2 Bifunctional enzyme   ?
fructose-6-phosphate ATP ?
fructose-2,6-bisphosphate ? fructose-2,6-bisphos
phate ? fructose-6-phosphate
Pi Enzyme highly regulated to control levels of
F 2,6-P
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PHOSPHOFRUCTOKINASE-2/ FRUCTOSE
BISPHOSPHATASE-2 ATP F-6-P
PFK- 2 FBPase-2 Pi   ADP
F2,6 bisphosphate   The next slides show how
these activities are controlled
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increases F 2,6 BP PFK-2 FBPase-2 single
protein-active inactive two functions
ATP
glucagon
PKA (?cAMP) P
ADP PFK-2 FBPase-2 inactive
active PKA protein decreases F 2,6
BP kinase A ?glycolysis, ?gluconeogenesis
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Glucagon hormone released when glucose levels
are low. ? signal to elevate blood glucose
levels ? increases intracellular levels of cAMP
in liver and elsewhere ? cAMP activates protein
kinase A cAMP-dependent protein kinase ?
stimulating gluconeogenesis and glycogenolysis
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Consequences A. glucose? AMP? ?
?PFK-2? ?F2,6bisP? ?PFK-1? ?glycolysis Also ?F1,6
BPase ??gluconeogenesis B. glucose? ATP? ?
?cAMP ? ?FBPase-2 ? ?F2,6 bisP? ?F1,6
BPase??gluconeogenesis Also ?PFK-1? ?glycolysis
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• Summary of Gluconeogenesis
• ? purpose- alternative source of
• glucose rather than dietary
• carbohydrates or glycogen breakdown
• primary precursors are pyruvate,
• lactate, glycerol, part of fatty acids
• and certain amino acids
• (glucogenic)
• ? 3 essentially irreversible steps
• of glycolysis are bypassed

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• ? regulated via pyruvate carboxylase,
• fructose 1,6 bisphosphatase, and
• phosphofructokinase-2/fructose
• bisphosphatase-2
• Notice glucose cannot be made
• from acetyl CoA

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GLYCOGEN METABOLISM   Glycogen a
highly branched polymer of glucose. Chains have
glycosidic links a 1?4. Branches are linked a 1?6.
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?Glucose stored in polymeric form as glycogen
mostly in the liver and skeletal muscle.   ?
Glucose can be rapidly delivered to the blood
stream when needed upon degradation of
glycogen. glycogenolysis ? Enough glucose and
energy triggers synthesis of glycogen.
glycogenesis
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• Glycogenesis
• GLYCOGEN BIOSYNTHESIS
• Glucose is transported into the liver
• cell by a specific glucose transporter
• and immediately phosphorylated.
• -- Most of the glucose in a cell is in
• the form of glucose-6-phosphate.

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1- Conversion of glucose-6-phosphate to
glucose-1-phosphate Enzyme phosphoglucomutase
a-D-glucose-6- a-D- glucose-1-
phosphate phosphate ? reversible reaction
allows G1P conversion to G6P in glycogenolysis ?
mechanism involves phosphorylated enzyme
intermediate and glucose-1,6 bisphosphate bound
intermediate similar to phosphoglycerate mutase
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2- Synthesis of Uridine
Diphosphoglucose Enzyme UDP-glucose
pyrophosphorylase Reaction
  glucose-1-phosphate UTP ?
PPi
Then PPi ? 2 Pi
UDP-glucose
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? the commiting step ? Phosphoryl transfer --
UTP is the energy equivalent of ATP -- energy is
used to activate glucose -- two phosphates from
UTP are lost as PPi ? PPi is broken down by an
enzyme PPi ? 2 Pi driving the reaction
to the right
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3- Glycogen synthesis Enzyme glycogen
synthase   UDP-glucose (glucose)n?UDP(glucose)n
1 ?
glycogen
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? glucose always added to nonreducing end. The
glycosidic bond formed is a (1 ? 4). ? glycogen
synthase is inhibited by phosphorylation,
regulated by glucagon ( discussed later)
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4- Branching Enzyme branching
enzyme   ? introduces branching by transferring
a teminal fragment of 6-7 residues from a
growing chain to a 6-position farther back in a
chain. ? makes a branch with an a (1?6)
link creating two ends to add glucose. ?
branching accelerates the rate of glucose release
during degradation.
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? ???? ?????????? ? ?
? new ? ?
? 1,6 ??? ? branching
? bond ? ? enzyme ? ? ?
? ? ? ? ?
? ? ?? ??
? ?
? ? ?
?
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-- GLYCOGENOLYSIS-- DEGRADATION OF
GLYCOGEN 1. Release of glucose-1-phosphate
Enzyme glycogen phosphorylase non- reducing
???????? Pi ends ????????????
glucose-1- ???? phosphate
?????????
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? always acts at nonreducing end ? 1,4 glycosidic
link is cleaved by phosphorylysis with retention
of energy potential in the phosphate ester of
glucose-1-phosphate.
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? stops at fourth glucose from a 1,6 branch
point ? contrast with enzymes acting on starch
and glycogen in the gut, which yield sugars, not
sugar phosphates, as products. ? activated by
phosphorylation, regulated by glucagon
and epinephrine
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2. Debranching - two parts Enzyme debranching
enzyme (both) ??? ? ?
(1?6) link ????????????
transferase
? ?????????????? Transfers chain of three
glucoses to any nonreducing end
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? ? (1?6)
link ??????????????
debranching enzyme
(glucosidase) ??????????????
? glucose 1,6
linkage cleaved
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?????????????? glycogen
phosphorylase or
phosphorylase for short
glucose-1-phosphate one at a
time as
previously shown -- phosphoglucomutase then
yields glucose-6-phosphate, which can be
dephosphorylated or enter glycolysis.
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Metabolic Regulation of Mammalian Glycogen
Levels   -- Glycogen reserves are the
most immediately available large source
of metabolic energy for mammals. -- Storage and
utilization are under dietary and hormonal
control.
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? Primary hormones -- epinephrine (adrenaline)

fight-or-flight -- glucagon -- insulin   ?
Primary enzyme targets in glycogen
metabolism glycogen phosphorylase and glycogen
synthase. The actions of the hormones are
indirect.
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Example- hormones and diet   Dinner at 900 pm --
steak, mashed potatoes, sherbert for dessert,
wine   Sleep immediately, sleep late   During
sleep amino acids, CH2O ? high blood
glucose levels ? higher insulin ?
glycogenesis
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Wake late for class??adrenaline rush
? run to class glycogen ? glucose ?
lactate epinephrine ( adrenaline) ?
glycogenolysis
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HORMONES Glucagon - low
glucose levels -- A polypeptide hormone
produced in a-cells of the islets of Langerhan of
the pancreas. -- Acts primarily on liver
cells. -- Receptors on surface of liver cells. --
Stimulates glycogen breakdown inhibits
glycogenesis. -- Glucagon also blocks
glycolysis stimulates gluconeogenesis.
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Epinephrine - low glucose levels   -- Acts
primarily on skeletal muscle. -- Receptors on
surface of cells. -- Stimulates glycogen
breakdown inhibits glycogenesis. Glucagon and
epinephrine both stimulate intracellular pathway
via increasing levels of cAMP.
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Insulin -- High levels of glucose induce
release of insulin from ß-cells of islets
of Langerhan in the pancreas. -- Insulin is
polypeptide hormone. -- Detected by receptors at
surface of muscle cells. -- Increases
glycogenesis in muscle. -- Intracellular signal
pathway involves complex sequential
phosphorylations and dephosphorylations.
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cAMP Cascade   -- A cyclic AMP cascade is used by
both epinephrine and glucagon. -- A cascade is
a mechanism in which enzymes activate other
enzymes sequentially usually leading to an
amplification of an initial signal.
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Epinephrine/Glucagon Cascade Regulating Glycogen
Metabolism
Epinephrine
Outside
Receptor
PM
AC
G- Protein
Inside
ATP cAMPPPi
AC adenylate cyclase
PM plasma membrane
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cAMP binds to protein kinase A,
activating it R R cAMP RR
C C C C cAMP cAMP
tetramer dimer 2 active

monomers R2C2 inactive R2 2C
active Dissociation caused by conformational chang
e induced by binding of cAMP Active kinase C
subunits add phosphate to proteins using ATP
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Catalytic C subunit phosphorylates phosphorylase
kinase, activating it
C monomer   ATP phosphorylase kinase
? phosphorylase kinase-P ADP
active Note subunit C also phosphorylates glyc
ogen synthase, inactivating it
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Active phosphorylase kinase transfers phosphate
to glycogen phosphorylase, activating this
enzyme. ATP phosphorylase b ? ADP
phosphorylase a- P
Glycogen
phosphorylase a (active form) then degrades
glycogen glycogen Pi ? glucose-1-P(glucose)n-
1
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How is Glycogenolysis Inhibited? -- when need
for glucose is over, glycogenolysis is
inhibited. -- a phosphoprotein phosphatase hydroly
zes the protein phosphates to reconvert phosphory
lase a ? phosphorylase b (inactive)
and phosphorylase kinase (active) ?
phosphorylase kinase
(inactive)
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How is Glycogenesis Activated? -- Complex process
stimulated by insulin. -- Insulin indirectly
activates a phosphoprotein phosphatase glycogen
synthase(D)?glycogen synthase(I) phosphorylated
dephosphorylated less active
active dependent on G-6-P independent of
G-6-P
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-- dephosphorylation is the major pathway for
stimulation of glycogenesis in liver and resting
muscles. -- Another way In active muscle, there
may still be high glucose-6-phosphate. The less
active phosphorylated glycogen synthase can be
activated by high levels of glucose-6-phosphate. -
- bypasses hormonal regulation
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How is Glycogenesis Inhibited?  -- epinephrine
and glucagon inhibit glycogen synthesis. a)
protein kinase A subunit C phosphorylates
glycogen synthase, decreasing its activity. b)
also phosphorylase kinase can phosphorylate
glycogen synthase, inactivating it. Its called
synthase phosphorylase kinase because of its dual
function.
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SIMPLISTIC SUMMARY -- Epinephrine and
glucagon stimulate glycogenolysis and inhibit
glycogenesis via a cAMP and a phosphorylation casc
ade. ? releases glucose -- Glycogenesis is
stimulated by insulin in a pathway ending in
the dephosphorylation of glycogen synthase. --
Glycogenolysis is also inhibited via
dephosphorylation. ? takes up glucose
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Glycogen Storage Diseases   -- A family of
serious, although not necessarily fatal, diseases
caused by mutations in the enzymes involving in
glycogen storage and breakdown.
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