GLUCONEOGENESIS
Formation of glucose from
noncarbohydrate sources
1
The source of pyruvate and oxaloacetate for
gluconeogenesis during fasting or carbohydrate
starvation is mainly amino acid catabolism.
Some amino acids are catabolized to pyruvate,
oxaloacetate, or precursors of these.
Muscle proteins may break down to supply amino acids.
These are transported to liver where they are
deaminated and converted to gluconeogenesis inputs.
Glycerol, derived from hydrolysis of triacylglycerols in fat
cells, is also a significant input to gluconeogenesis.
2
Noncarbohydrate precursors of
glucose
Triglycerols
Fatty acids
glycerol
Dietary & muscle
proteins
Amino acids
3
Main sites of gluconeogenesis:
• Major site: Liver.
• Minor site: Kidney.
• Very little:
– Brain.
– Muscle (skeletal and heart).
In liver and kidney it helps to maintain the
glucose level in the blood so that brain and
muscle can extract sufficient glucose from it to
meet their metabolic demands.
4
Gluconeogenesis Versus Glycolysis:
• 7 steps are shared between glycolysis and
gluconeogenesis.
• 3 essentially irreversible steps shift the
equilibrium far on the side of glycolysis.
• Most of the decrease in free energy
(consuming energy) in glycolysis takes place
during these 3 steps.
5
6
In gluconeogenesis the three reactions are
bypassed by a set of separate enzymes.
1. Phosphoenolpyruvate is formed from
pyruvate:
2. Fructose 6-phosphate is formed from fructose 1,6bisphosphate:
3. Glucose is formed by hydrolysis of glucose 6-phosphate:
7
Bypass of Pyruvate Kinase:
Pyruvate Kinase (last step of Glycolysis) catalyzes:
phosphoenolpyruvate + ADP  pyruvate + ATP
For bypass of the Pyruvate Kinase reaction, cleavage of 2
~P bonds is required.
 DG for cleavage of one ~P bond of ATP is insufficient
to drive synthesis of phosphoenolpyruvate (PEP).
 PEP has a higher negative DG of phosphate hydrolysis
than ATP.
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PYRUVATE
PHOSPHOENOLPYRUVATE
2 Enzymes needed to bypass pyruvate kinase
A
B
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Cytosolic PEP
CARBOXYKINASE
Oxaloacetate
Cytosolic Malate
DEHYDROGENASE
NADH
PEP
+ H+
CO2
NAD+
Malate
Malate
NAD+
H+ +
NADH
Oxaloacetate
Pyruvate
CARBOXYLASE
CO2
Pyruvate
Pyruvate
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Cytosolic PEP
CARBOXYKINASE
Oxaloacetate
Cytosolic Malate
DEHYDROGENASE
NADH
PEP
+ H+
CO2
NAD+
Malate
PEP
Malate
NAD+
H+ +
NADH
CO2
Oxaloacetate
Oxaloacetate
Pyruvate
CARBOXYLASE
Mitochondrial PEP
CARBOXYKINASE
CO2
Pyruvate
CARBOXYLASE
CO2
Pyruvate
Pyruvate
Pyruvate
NADH
Pyruvate
+ H+
NAD+
Lactate DEHYDROGENASE
Lactate
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• This pathway predominates when lactate is the
precursor.
• The conversion of lactate to pyruvate in the
hepatocyte cytosol yields NADH.
• Thus no Malate transport is needed any more for
this purpose.
• The mitochondrial and cytosolic PEP
CARBOXYKINASE enzymes are encoded by
separate nuclear genes. (two different enzymes
catalyzing the same reaction in different
localizations)
12
• To keep glucose inside the cell, the generation
of free glucose is controlled in two ways:
1.The enzyme responsible for the conversion of
glucose 6-phosphate into glucose, glucose 6phosphatase, is regulated.
2.The enzyme is present only in tissues whose
metabolic duty is to maintain blood-glucose
balanced by releasing glucose into the blood (the
liver and to a lesser extent the kidney).
13
• Several endoplasmic
reticulum (ER) proteins play
a role in the generation of
glucose from glucose 6phosphate.
– T1 transports glucose 6-phosphate
into the lumen of the ER.
– T2 and T3 transport Pi and glucose,
respectively, back into the cytosol.
– Glucose 6-phosphatase is stabilized
by a Ca2+-binding protein (SP).
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REGULATION OF CARBOHYDRATE
METABOLISM
15
• The rate of conversion of glucose into
pyruvate is regulated to meet two
major cellular needs:
1. The production of ATP, generated by the
degradation of glucose.
2. The provision of building blocks for
synthetic reactions, such as the formation
of fatty acids.
16
Regulation and control of enzyme
activity
•
•
•
•
Substrate level control.
Allosteric effectors
Covalent modification
Enzyme concentration:
1. increased synthesis
2. generation of active enzyme by processing
• Substrate cycles
17
Substrate level control
• Since most often [S] >
Km, the change in
substrate
concentration does not
change the reaction
rate appreciably.
• Thus, controlling a
metabolic flux is not
normally achieved by
varying substrate
concentrations.
18
Allosteric effectors
• Noncovalently bind
and regulate the
enzyme.
• The effector may be
stimulatory or
inhibitory.
• The substrate and
effector usually
occupy different
specific binding sites.
19
Allosteric enzymes kinetics:
• Sigmoid kinetic
behavior is seen.
• K0.5 represents the
substrate
concentration at which
the enzyme velocity is
half Vmax.
• (-) and (+) respectively
indicate inhibitory and
stimulatory effectors.
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Covalent modification
21
Enzyme concentration: increased
synthesis
22
Enzyme concentration: generation of
active enzyme by processing
23
Glycolysis & Gluconeogenesis are both spontaneous.
If both pathways were simultaneously active in a cell, it
would constitute a "futile cycle" that would waste energy.
Glycolysis:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
Gluconeogenesis:
2 pyruvate + 2 NADH + 4 ATP + 2 GTP 
glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
Questions:
1. Glycolysis yields how many ~P ? 2
2. Gluconeogenesis expends how many ~P ? 6
3. A futile cycle of both pathways would waste how many
~P per cycle ? 4
24
Gluconeogenesis and Glycolysis Are
Reciprocally Regulated
• The amounts and activities of the distinctive
enzymes of each pathway are controlled so
that both pathways are not highly active at
the same time.
• The interconversion of fructose 6-phosphate
and fructose 1,6-bisphosphate is stringently
controlled.
– Phosphofructokinase and fructose 1,6bisphosphatase are reciprocally controlled by
fructose 2,6-bisphosphate in the liver
25
26
ALLOSTERIC REGULATORS OF PFK-1
and FBPase-1
27
PHOSPHOFRUCTOKINASE:
The most important control element in the mammalian
glycolytic pathway.
• PFK in the liver is a
tetramer of 4
identical subunits.
• The allosteric
effectors binding site
is distinct from the
catalytic site.
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ATP allosterically inhibit the enzyme:
• High concentrations of
ATP converts the
hyperbolic binding
curve of F6-P to
sigmoidal one.
• AMP reverses the
inhibitory effect of ATP
– The activity of the enzyme
increases when the ATP/AMP
ratio is lowered
glycolysis is stimulated
as the energy charge falls
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AMP but not ADP positive regulator of
PFK-1
• When ATP is utilized rapidly, the enzyme
Adenylate Kinase forms ATP and AMP from
ADP:
ADP + ADP
ATP + AMP
• AMP becomes the signal for low energy
charge.
30
Citrate inhibits PFK-1 enzyme
• A high level of citrate means that biosynthetic
precursors are abundant and additional
glucose should not be degraded for this
purpose.
• Citrate inhibits PFK-1 by enhancing the
inhibitory effect of ATP.
31
F2,6-BP allosterically activates PFK-1
and inhibits FBPase:
• Phosphofructokinase and fructose 1,6bisphosphatase are reciprocally controlled by
fructose 2,6-bisphosphate in the liver.
32
How is the concentration of F 2,6-BP
appropriately controlled?
• F2,6-BP is formed in a reaction catalyzed by
Phosphofructokinase-2 (PFK-2)
• It is hydrolyzed to F6-P by Fructose
Bisphosphatase-2 (FBPase-2)
• Both PFK-2 and FBPase-2 are part of the same
55Kd polypeptide chain.
• The bifunctional enzyme
Exists in 5 isozymic forms.
– L-isoform in liver.
– M-isoform in muscle.
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Why is phosphofructokinase rather
than hexokinase the pacemaker of
glycolysis?
• Glucose 6-phosphate is not solely a glycolytic
intermediate.
– It can also be converted into glycogen or it can be oxidized by
the pentose phosphate pathway to form NADPH.
• The first irreversible reaction unique to the
glycolytic pathway, the committed step, is the
phosphorylation of fructose 6-phosphate to
fructose 1,6-bisphosphate.
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The interconversion of PEP and pyruvate is
precisely regulated.
• gluconeogenesis is favored when the cell is rich in
biosynthetic precursors and ATP.
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GLUCOSE TRANSPORTERS
36
HORMONES control the amount and
activities of essential enzymes
• Hormones affect gene expression primarily
by:
1. changing the rate of transcription
2. regulating the degradation of mRNA.
• Transcriptional control in eukaryotes is much
slower than allosteric control;
– it takes hours or days in contrast with seconds to
minutes.
37
Insulin
• Rises subsequent to eating and
stimulates the expression of:
– Phosphofructokinase
– pyruvate kinase
– PFK-2/FBPase-2
38
Glucagon
• Rises during starvation:
• inhibits the expression of:
– Phosphofructokinase
– pyruvate kinase
– PFK-2/FBPase-2.
• stimulates instead the production of two key
gluconeogenic enzymes:
– phosphoenolpyruvate carboxykinase
– fructose 1,6-bisphosphatase
39
Hormones work at the promoter level
• The PEP-Carboxykinase promoter approximately 500 bp in length
• Contains regulatory sequences (response elements) that mediate
the action of several hormones:
–
–
–
–
IRE: insulin response element
GRE: glucocorticoid response element
TRE: thyroid hormone response element
CREI and CREII: cAMP response elements.
40
Substrate Cycles:
• Both reactions are not
simultaneously fully active in most
cells, because of reciprocal allosteric
controls.
ATP
F 6-P
Pi
F1,6-BPase
PFK-1
H2O
ADP
F 1,6-BP
41
Substrate cycles are biologically
important:
1. Substrate cycles amplify
metabolic signals:
– This amplification is made possible by
the rapid hydrolysis of ATP.
If an allosteric effector reciprocally increases A to B and
decreases B to A by 20% each
Then a 20% change in the rates of the opposing
reactions has led to a 480% (=100x48/10) increase in
the net flux.
2. generation of heat produced by
the hydrolysis of ATP.
42
Lactate produced by active skeletal muscle and
erythrocytes is a source of energy for other
organs.
• The only purpose of the reduction of pyruvate to
lactate is to regenerate NAD+ so that glycolysis
can proceed in active skeletal muscle and
erythrocytes.
• lactate is a dead end in metabolism.
– It must be converted back into pyruvate before it can be
metabolized.
43
The Cori Cycle operates during exercise.
For a brief burst of ATP utilization, muscle cells utilize ~P
stored as phosphocreatine.
Once phosphocreatine is exhausted, ATP is provided
mainly by Glycolysis, with the input coming from
glycogen breakdown and from glucose uptake from the
blood.
(Aerobic fat metabolism, discussed elsewhere, is more
significant during a lengthy period of exertion such as a
marathon run.)
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Cori Cycle
Liver
Glucose
2 NAD+
2 NADH
6 ~P
2 Pyruvate
2 NADH
2 NAD+
2 Lactate
Blood
Muscle
Glucose
2 NAD+
2 NADH
2 ~P
2 Pyruvate
2 NADH
2 NAD+
2 Lactate
Lactate produced from pyruvate passes via the blood to
the liver, where it may be converted to glucose.
The glucose may travel back to the muscle to fuel
Glycolysis.
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Cori Cycle
Liver
Glucose
2 NAD+
2 NADH
6 ~P
2 Pyruvate
2 NADH
2 NAD+
2 Lactate
Blood
Muscle
Glucose
2 NAD+
2 NADH
2 ~P
2 Pyruvate
2 NADH
2 NAD+
2 Lactate
The Cori cycle costs 6 ~P in liver for every 2 ~P made
available in muscle. The net cost is 4 ~P.
Although costly in ~P bonds, the Cori Cycle allows the
organism to accommodate to large fluctuations in energy
needs of skeletal muscle between rest and exercise. 46
The equivalent of the Cori Cycle also operates during
cancer.
If blood vessel development does not keep pace with
growth of a solid tumor, decreased O2 concentration
within the tumor leads to activation of signal processes
that result in a shift to anaerobic metabolism.
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Liver
Glucose
2 NAD+
2 NADH
6 ~P
2 Pyruvate
2 NADH
2 NAD+
2 Lactate
Blood
Cancer Cell
Glucose
2 NAD+
2 NADH
2 ~P
2 Pyruvate
2 NADH
2 NAD+
2 Lactate
Energy dissipation by the Cori Cycle, which expends 6
~P in liver for every 2 ~P produced via Glycolysis for
utilization within the tumor, is thought to contribute to
the weight loss that typically occurs in late-stage cancer
even when food intake remains normal.
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