Gluconeogenesis

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• Gluconeogenesis: The synthesis of glucose from non carbohydrate precursors
• Major substrates are: glucogenic amino acids, lactate, glycerol, and propionate
• Liver and kidney are the major gluconeogenic tissues
• Meets needs of the body for glucose when carbohydrates is not available in
sufficient amount from the diet or glycogen reserves
• A supply of glucose is necessary because the brain and erythrocytes are highly
dependent on glucose as the primary fuel
• A typical adult brain requires about 120g glucose per day, which accounts for most
of the 160g of glucose needed by the whole body
• Gluconeogenesis also -maintains levels of CAC intermediates
-clears lactate produced by skeletal
muscles and red blood cells
-clears glycerol produced by adipose tissue
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 Glucose ATP
Pi
Glycogen
Glucose 6-phosphate ADP
Gluconeogenesis
pathway
Pi Fructose 6-phosphate ATP
– +
AMP, Fructose 2, 6- ADP Fructose 2, 6-
Fructose 1, 6-bisphosphate
bisphosphate bisphosphate
Dihydroxyacetone G 3- P
Glyceraldehyde 3- NADH dehydro
phosphate
phosphate NAD+ genase
NAD+
NAD+ NADH Glycerol 3- P
Glycerol
NADH
NADH 1,ADP
3-bisphosphoglycerate Glycerol kinase
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ADP
NAD+ GTP GDP ATP ATP
Oxaloacetate 3-phosphoglycerate
Malate
CO2 2-phosphoglycerate
Malate
NAD+ Phosphoenolpyruvate
ADP ATP ADP Glycolytic
NADH
pathway
Oxaloacetate Pyruvate Pyruvate ATP
Acetyl-CoA +
CO2 Alanine Mitochondria
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Alanine
Thermodynamic barriers prevent reversal of glycolysis
• Three nonequilibrium reactions catalysed by glucokinase/hexokinase, phosphofructokinase and pyruvate kinase prevent
reversal of glycolysis for gluconeogenesis
These are bypassed by the following new steps:
➢ Conversion of pyruvate to phosphoenolpyruvate
• Pyruvate is first transported from the cytosol into the mitochondria or generated from alanine within the mitochondria by
transamination
• Mitochondrial pyruvate carboxylase catalyze the carboxylation of pyruvate to oxaloacetate at an expense of an ATP.
• Biotin which carries activated CO2 is the coenzyme for the reaction
• Pyruvate carboxylase is a mitochondrial enzyme whereas other enzymes of gluconeogenesis leading to glucose formation are
cytosol
• Oxaloacetate is reduced to malate in the mitochondria by an NADH-linked malate dehydrogenase
• Malate leaves the mitochondria through the malate—α-ketoglutarate transporter of the inner mitochondrial membrane
• In the cytosol malate is oxidized to oxaloacetate by NAD+ linked malate dehydrogenase in the cytosol with the production of
cytosolic NADH
• Phosphoenolpyruvate carboxykinase catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate
at an expense of a second high-energy phosphate (GTP as phosphate donor). Source of GTP is succinyl-CoA synthetase in the
CAC
• Two high energy phosphate equivalents are expended to phosphorylate one molecule of pyruvate to phosphoenolpyruvate
• The NADH generated by oxidation of malate is consumed in gluconeogenesis (conversion of 1,3-bisphosphoglycerate to
glyceraldehyde 3-phosphate) 4
• A second (and shorter pass) predominates when lactate is the predominant
glucogenic precursor
• Conversion of lactate to pyruvate in the hepatocyte cytosol yields NADH and
the export of reducing equivalents from the mitochondria is therefore
unnecessary
• Pyruvate produced by lactate dehydrogenase then enters the mitochondria
where its converted to oxaloacetate by pyruvate carboxylase

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• Oxaloacetate is then converted directly to phosphoenolpyruvate by a
mitochondrial isoenzyme of phosphoenolpyruvate carboxykinase
• Phosphoenolpyruvate is then transported out of the mitochondria to continue on
the gluconeogenic path
• Reversal of the reaction catalysed by pyruvate kinase involves two exergonic
reactions

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➢ Conversion of fructose 1,6 bisphosphate to fructose 6-phosphate is the second bypass
• The reversal of the reaction catalyzed by phosphofructokinase is effected by fructose 1,6 bisphosphatase.
• Mg2+ dependent
• Promotes the irreversible hydrolysis of the C-1 phosphate (not phosphoryl group transfer to ADP)
Fructose 1,6-bisphosphate + H2O Fructose 6-phosphate + Pi

➢ Conversion of glucose 6-phosphate to free glucose is the third pass

• Catalyzed by Mg2+ activated glucose 6-phosphatase
• As above promotes the irreversible hydrolysis of the C-6 phosphate (not phosphoryl group transfer to ADP)
Glucose 6-phosphate + H2O Glucose + Pi
• Glucose 6-phosphatase is found in hepatocytes and renal cells. It is not present in brain and muscle

Gluconeogenesis is expensive
• For each molecule of glucose formed from pyruvate, six high energy phosphate groups are required; four from ATP and two from
GTP
• In addition two molecules of NADH are required for the reduction of two molecules of 1,3-bisphosphoglycerate
• Much of the energy is required to ensure irreversibility of gluconeogenesis

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Citric acid intermediates and many amino acids are gluconeogenic
• All citric acid intermediates are gluconeogenic as they can undergo oxidation to oxaloacetate
• After transamination and deamination ,glucogenic amino acids yield either pyruvate or CAC intermediates

• Alanine and glutamine the principal molecules used to transport amino groups from extrahepatic tissues to the liver are
particularly important gluconeogenic amino acids.
• After removal of their amino groups in the liver, the carbon skeletons remaining (pyruvate and α-ketoglutarate
respectively) are funneled into gluconeogenesis
• No conversion of fatty acids to glucose occurs in mammals
• Even-number fatty acids yield only acetyl-CoA on oxidative cleavage
• Acetyl-CoA can not be a precursor to glucose; the pyruvate dehydrogenase reaction is irreversible and no other
pathway exists for the conversion of acetyl-CoA to pyruvate

Glycerol
• Is released from adipose tissue as a result of lipolysis
• Glycerol kinase catalyzes the conversion of glycerol to glycerol 3-phosphate
• Glycerol 3-phosphate may be oxidized to dihydroxyacetone phosphate by NAD+ in a reaction catalyzed by glycerol 3-
phosphate dehydrogenase

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Serine, Cysteine,
Threonine, Glycine,
Hydroxyproline
Lactate

Tryptophan Transaminase
Alanine
Pyruvate
Pyruvate Carboxylase
Glucose Phosphoe Oxaloacetate
nolpyruva
te Transaminase
Aspartate
Tyrosine, Fumarate
Phenylalanine Citrate

Isoleucine,
Methionine,
Valine Succinyl-CoA

Propionate
α-Ketoglutarete
Histidine,
Proline, Transaminase
Glutamine,
Arginine Glutamate 8
Futile cycles in carbohydrate metabolism consume ATP
• At each of the point where glycolytic reactions are bypassed by gluconeogenic reactions simultaneous
operation of both pathways would be wasteful
eg Phosphofructokinase-1 and fructose1,6-bisphosphatase catalyze opposing reactions
ATP + fructose 6-phosphate → ADP + fructose 1,6-bisphosphate
Fructose 1,6-bisphosphate + H2O → fructose 6-phosphate + Pi

The sum of the two reactions is

ATP + H2O → ADP + Pi + heat
A large amount of energy would be wasted as heat
• This uneconomical cycle is known as a futile cycle
• Under normal circumstances futile cycling does not occur at a significant rate, because it is prevented by
reciprocal regulatory mechanism

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 Gluconeogenesis and glycolysis are reciprocally controlled

Glycolysis Gluconeogenesis

Fructose 6-phosphate
F-2,6-BP, AMP Fructose 1,6 F-2,6-BP, AMP
+ Phosphofructo- bis- –
ATP, Citrate, H+ kinase-1 phosphatase Citrate
– +
Fructose 1,6-
bisphosphate

Several steps

Phosphoenolpyruvate
ADP
Phosphoenolpyruvate –
carboxykinase
F 1,6-BP
+ Pyruvate Oxaloacetate
kinase Pyruvate
ATP, Alanine
– carboxyl
ase Acetyl-CoA
Pyruvate +
– ADP
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• Gluconeogenesis and glycolysis are coordinated so that one pathway is relatively inactive while
the other is highly active.
• The rate of glycolysis is also determined by the concentration of glucose and the rate of
gluconeogenesis by the concentration of lactate and other precursors of glucose
The interconversion of fructose 6-phosphate and fructose1,6 bisphosphate is stringently
controlled
• Phosphofructokinase -1 is - stimulated by AMP, fructose 2,6 bisphosphate (F2,6-BP, increases
enzyme affinity for fructose 6- phosphate and diminishes the inhibitory effect of ATP).
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-inhibited by ATP and citrate
• Fructose 1,6 bisphosphatase is -inhibited by AMP, F2,6-BP
-activated by citrate
• A high level of AMP indicates that energy charge is low and signals the need for ATP generation.
A high level of ATP and citrate indicates that the energy charge is high and biosynthetic
intermediates are abundant- under these conditions glycolysis is nearly switched off and
gluconeogenesis promoted

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Fructose 2,6-bisphosphatase plays a unique role in the regulation of glycolysis
and gluconeogenesis in the liver
• The most important effector of phosphofructokinase-1 and inhibitor of fructose
1,6-bisphosphatase in the liver is fructose 2,6 bisphosphate
• fructose 2,6 bisphosphate is formed by phosphorylation of fructose 6-phosphate by
phosphofructokinase-2. The same enzyme protein is also responsible for its breakdown since
it has fructose 2,6 bisphosphatase activity.
• When glucose is abundant the concentration of fructose 2,6 bisphosphate
increases, stimulating glycolysis by activating phosphofructokinase-1 and
inhibiting fructose1,6-bisphosphstase
• When glucose level is low glucagon stimulates the production of cAMP,
activating cAMP-dependent protein kinase, which in turn inactivates
phosphofructokinase-2 and activates fructose 2,6 bisphosphatase
• Gluconeogenesis is stimulated by a decrease in the concentration of fructose 2,6 bisphosphate
, which deactivates phosphofructokinase-1 and deinhibits fructose 1,6-bisphosphatase .

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The interconversion of phosphoenolpyruvate and pyruvate is also stringently
controlled
• Pyruvate kinase is controlled by allosteric effectors and phosphorylation
-inhibited by high levels of ATP and alanine which signal that the
energy charge is high and that the building block are abundant
-its also switched off when glucose is more urgently needed by brain
or muscle. cAMP dependent cascade leads to phosphorylation and consequent
inhibition of pyruvate kinase during starvation
-activated by fructose 1,6 bisphosphate to enable it to keep pace with
the oncoming high flux of intermediates.
• Pyruvate carboxylase is activated by acetyl-CoA and inhibited by ADP
• Phosphoenolpyruvate carboxykinase is inhibited by ADP when the energy charge is
low
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Hormone control affects gene expression primarily by changing the
rate of transcription
Transcriptional control occurs in hours or days where as allosteric
control in seconds to mins.
Insulin which rises following feeding stimulates formation of
phosphofructokinase-1, pyruvate kinase and the bifunctional enzyme
that makes and degrades F-2,6-BP
Glucagon which rises during starvation, inhibits the expression of these
enzymes and instead promotes expression of key gluconeogenic
enzymes phosphoenolpyruvate carboxykinase and fructose 1,6-
bisphosphatase.

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Lactate and alanine formed by the contracting muscle are converted into glucose
by the liver
The major raw materials of gluconeogenesis are lactate and alanine produced by
skeletal muscles and red blood cells
The rate of production of pyruvate by glycolysis exceeds the rate of oxidation of
pyruvate in the CAC in contracting muscle under anaerobic conditions, as during
vigorous exercise.
-under the same conditions the rate of formation of NADH by glycolysis is greater
than the rate of its oxidation by the respiratory chain
- continued glycolysis depends on the availability of NAD+ for the oxidation of
glyceraldehyde 3-phosphate
-the accumulation of pyruvate and NADH is reversed by lactate dehydrogenase,
which oxidizes NADH to NAD+, as it reduces pyruvate to lactate

NADH + H+ NAD+
Pyruvate Lactate

Lactateofdehydrogenase
The only purpose the reduction of pyruvate to lactate is to generate NAD+ so that
glycolysis can proceed in active skeletal muscle and erythrocytes

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• Lactate and pyruvate diffuse out of the skeletal muscles into the blood and are carried to the liver. Much more
lactate is carried because of the high NADH/NAD+ ratio in the contracting skeletal muscle
• In the liver lactate is oxidized to pyruvate , a reaction favoured by the low NADH/NAD+ ratio in the cytosol of
liver cells
• The liver furnishes glucose to contracting skeletal muscle, which derives ATP from the glycolytic conversion of
glucose to lactate. Glucose is then re synthesized from lactate by the liver. These reactions constitute the Cori
cycle
• Recent evidence suggests that alanine like lactate is a major precursor of glucose. In muscle alanine is formed
from pyruvate by transamination; the reverse reaction occurs in the liver
Effect of alcohol on gluconeogenesis
• Alcohol is primarily metabolized in the liver
• The 2 –step metabolism of alcohol leads to a rapid increase in hepatic NADH
alcohol dehydrogenase CH3CH2OH + NAD+ + H+ → CH3CHO + NADH
aldehydwe dehydrogenase CH3CHO + NAD+ + H+→ CH3COOH + NADH
• The increase in hepatic NADH shiftsthe equilibrium of lactate dehydrogenase reaction towards lactate formation,
limiting gluconeogenesisfrom pyruvate derived from lactate (or alanine)
• Shifts cytosolic oxaloacetate toward malate reducing gluconeogenesis from citric acid cycle intermediates
• Shifts dihydroxyacetone phosphate toward glycerol 3-phosphate limiting gluconeogenesis from glycerol

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 The Cori cycle

Liver Muscle

Glucose Glucose
Glycolysis
Gluconeogenesis

Pyruvate Pyruvate

Lactate Lactate

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En