GLUCONEOGENESIS

SHARI R. BABU
[email protected]
DEPT. OF PHYSIOLOGICAL SCIENCES
SCHOOL OF MEDICINE, UNZA

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 OVERVIEW OF GLUCONEOGENESIS

• Some tissues, such as the brain, red blood cells, kidney medulla, lens and cornea of
the eye, testes, and exercising muscle, require a continuous supply of glucose as a
metabolic fuel.

• Liver glycogen, an essential postprandial source of glucose, can meet these needs
for only 10–18 hours in the absence of dietary intake of carbohydrate.

• The brain uses almost 70% of the total glucose produced by liver during normal
fasting.

• During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose
is formed from non-carbohydrate precursors.

• Gluconeogenic precursors include intermediates of glycolysis and the tricarboxylic

acid (TCA) cycle, as well as glycerol, lactate, and the a-keto acids obtained from the
transamination of glucogenic amino acids.

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ALANINE LACTATE OXALOACETATE

Aspartate, α-
Ketoglutarate,
PYRUVATE Fumarate, Succinyl-CoA

GLUCONEOGENESIS
GLUCOGENIC
AMINO ACIDS

DIHYDROXYACETONE PHOSPHATE

GLYCEROL FATTY ACID

TRIACYLGLYCEROL

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• The formation of glucose does not occur by a simple reversal of glycolysis.

• Instead, glucose is synthesized by a special pathway that requires both

mitochondrial and cytosolic enzymes.

• During an overnight fast, approximately 90% of gluconeogenesis occurs in the

liver, with the kidneys providing 10% of the newly synthesized glucose
molecules.

• However, during prolonged fasting, the kidneys become major glucose-

producing organs, contributing an estimated 40% of the total glucose
production.

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REACTIONS UNIQUE TO GLUCONEOGENESIS

• Seven out of the ten glycolytic reactions are reversible and use the same
enzymes in the synthesis of glucose from pyruvate via gluconeogenesis.

• However, three of the glycolytic reactions are irreversible and must be

circumvented by four alternate reactions that energetically favors the synthesis
of glucose.

• The three irreversible reactions that needs to be circumvent are the glycolytic
reactions catalyzed by hexokinase, phosphofructokinase-1 and pyruvate
kinase.

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 Pink arrows Blue arrows indicate
indicate glycolytic the gluconeogenic
pathway pathway

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FORMATION OF PHOSPHOENOLPYRUVATE (PEP) FROM PYRUVATE

• Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves

two reactions.

1. Carboxylation of pyruvate: Pyruvate Carboxylase catalyzes an ATP-

requiring reaction in which the vitamin biotin is the coenzyme:

Pyruvate + HCO3- + ATP  Oxaloacetate + ADP + Pi

• Pyruvate carboxylase is allosterically activated by acetyl CoA and inhibited by

ADP.

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Transport of oxaloacetate (OAA) to the cytosol

• OAA must be converted to PEP for gluconeogenesis to continue.

• The enzyme that catalyzes this conversion is found in both the mitochondria
and the cytosol in humans.

• The PEP that is generated in the mitochondria is transported to the cytosol

by a specific transporter, whereas for PEP to form in the cytosol it requires
the transport of OAA from the mitochondria to the cytosol.

• OAA is unable to directly cross the inner mitochondrial membrane; it must

first be reduced to malate by mitochondrial malate dehydrogenase.

• Malate can be transported from the mitochondria to the cytosol, where it is

reoxidized to oxaloacetate by cytosolic malate dehydrogenase as NAD is +

reduced.

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CYTOSOL MITOCHONDRIAL
MATRIX

NAD+ NADH

MALATE OXALOACETATE
Malate
Dehydrogenase
Malate
Transporter
2. Decarboxylation of cytosolic oxaloacetate: Oxaloacetate is decarboxylated
and phosphorylated to PEP in the cytosol by PEP-carboxykinase. The reaction
is driven by hydrolysis of GTP.

Oxaloacetate + GTP  Phosphoenolpyruvate + GDP + CO2

• Then, PEP is acted on by the reactions of glycolysis running in the reverse

direction until it becomes fructose 1,6-bisphosphate.

• PEPCK is inhibited by ADP.

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DEPHOSPHORYLATION OF FRUCTOSE 1,6-BISPHOSPHATE

• Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase

bypasses the irreversible phosphofructokinase-1 reaction.

Fructose 1, 6-bisphosphate + H2O  Fructose 6-phosphate + Pi

• This reaction is an important regulatory site of gluconeogenesis.

• Fructose 1,6-bisphosphatase is inhibited by elevated levels of AMP while high

levels of ATP and citrate stimulate gluconeogenesis, an energy-requiring
pathway.

• Fructose 1,6-bisphosphatase, found in liver and kidney, is also inhibited by

fructose 2,6-bisphosphate.

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 SB7

• Fructose-2,6-bisphosphate is synthesized & degraded by a bi-functional

enzyme (PFK2/F2,6BPase) that includes 2 catalytic domains:

Phosphofructokinase-2 (PFK2) domain catalyzes:

Fructose-6-phosphate + ATP  Fructose-2,6-bisphosphate + ADP

Fructose-2, 6 Bisphosphatase (F2,6BPase) domain catalyzes:

Fructose-2,6-bisphosphate + H2O  Fructose-6-phosphate + Pi

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Slide 12

SB7 Phosphofructokinase 1 (PFK), which catalyses the phosphorylation of fructose-6-phosphate to

fructose-1,6- bisphosphate, is a key regulatory step in the glycolysis.

When glucose level is low, glucagon is released into the bloodstream, triggering a cAMP signal cascade.
In the liver Protein kinase A inactivates the PFK-2 domain of the bifunctional enzyme via phosphorylation.

The F-2,6-BPase domain is then activated which lowers fructose 2,6-bisphosphate (F-2,6-BP) levels.
Because F-2,6-BP normally stimulates phosphofructokinase-1(PFK1) and inhibits F-1,6BPase, the decrease
in its concentration leads to the inhibition of glycolysis and the stimulation of gluconeogenesis,
respectively.
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 GLUCAGON AND/OR EPINEPHRINE

cAMP-dependent Protein Kinase

(PKA)

+ Phosphorylation -

P
PFK-2 PFK-2

F-2,6-BPase F-2,6-BPase P

- +

Fructose-2, 6-Bisphosphate Fructose-6-Phosphate +

Pi

• Decreased levels of F-2,6-BP means inhibition of F-1,6-BPase is lifted.

• This increases the rate of Gluconeogenesis.
 INSULIN

Protein Phosphatase-1

Dephosphorylation
+ -
P

P
F-2,6-BPase F-2,6-BPase

P
PFK-2 PFK-2

P
- +

Fructose-6-Phosphate + ATP Fructose-2, 6-

Bisphosphate + ADP

• Increased levels of F-2,6-BP activates PFK-1.

• This increases the rate of Glycolysis.
In presence of glucagon or PKA action In presence of insulin,
epinephrine, cAMP levels glucose will enter cells.
are increased.

F2,6BPase The levels of fructose-6-

cAMP-dependent protein phosphate will increase.
kinase (PKA) is activated.
PFK2 Insulin decreases the
levels of cAMP.
PKA activates F2,6BPase
domain via
phosphorylation. Activate Insulin also activates
Inactive
protein phosphatase-1
(PP1).
Once activated,
F2,6BPase will breakdown PP1 dephosphorylates
F2,6BP and lift its inhibition and activates PFK2.
PP1 action
on F1,6BPase.

This results in increased

PKA inactivates PFK2 via levels of F26BP which
phosphorylation preventing activates PFK1.
formation of F2,6BP.
Also, PP1
dephosphorylates and
Gluconeogenesis inactivates F26BPase.
stimulated.

Glycolysis is stimulated.
SB8

DEPHOSPHORYLATION OF GLUCOSE 6-PHOSPHATE

• Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase bypasses the

irreversible hexokinase reaction.

Glucose 6-phosphate + H2O  Glucose + Pi

• Release of free glucose requires two proteins: glucose 6-phosphate

translocase, which transports glucose 6-phosphate across the ER membrane,
and the ER enzyme, glucose 6-phosphatase, which removes the phosphate,
producing free glucose.

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Slide 16

SB8 The deficiency of any one of the four unique gluconeogenic enzymes i.e., pyruvate carboxylase,
phoshoenolpyruvate carboxykinase, fructose 2,6-bisphosphatase and glucose-6-phosphatase, can lead to
hypoglycemia during periods of fasting. Especially overnight fasting when the liver glycogen reserves
have been depleted.
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SB9

HORMONAL REGULATION OF GLUCONEOGENESIS

• Glucagon lowers the level of fructose 2,6-bisphosphate, resulting in activation

of fructose 1,6-bisphosphatase and inhibition of PFK-1, thus favouring
gluconeogenesis.

• Glucagon elevates cAMP level and cAMP-dependent protein kinase activity,

which stimulates the conversion of pyruvate kinase to its inactive
(phosphorylated) form.

• This decreases the conversion of PEP to pyruvate, which has the effect of
diverting PEP to the synthesis of glucose.

• Glucagon increases the transcription of PEP-carboxykinase gene, increasing

the levels of the enzyme.

• Insulin increases the activity of phosphofructokinase-1, pyruvate kinase and

the levels of F-2,6-BP.

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Slide 17

SB9 So in the liver and kidneys, glycolysis and gluconeogenesis do not occur simultaneously.
But it is possible for gluconeogenesis to occur in the liver while glycolysis occurs in extra-hepatic tissues.
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CLINICAL SIGNIFICANCE

• In the absence of glucose-6-phosphatase, gluconeogenesis is impaired, and

this results in fasting hypoglycemia. This can occur in Von Gierke’s disease
when the enzyme is deficient.

• Diabetes is either the result of impaired insulin production or decreased

insulin sensitivity.
• Insulin is an inhibitor of gluconeogenesis. In the absence of insulin,
gluconeogenesis occurs at a rapid rate, exacerbating hyperglycemia.

• Alcoholism can induce hypoglycemia.

• Alcohol is oxidized to acetaldehyde and then to acetate in the liver. These
reactions increase the level of NADH.
• The high levels of NADH favors the formation of lactate from pyruvate.
• This lowers the level of pyruvate that can enter gluconeogenesis, which
causes hypoglycemia.
• As a result, heavy ethanol consumption can lead to both lactic acidosis
and hypoglycemia.
SB10

The Cori Cycle

• Lactate is formed by active skeletal muscle when the rate of glycolysis

exceeds the rate of oxidative metabolism.

• Lactate is readily converted into pyruvate by the action of lactate

dehydrogenase.

• During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate

by lactate dehydrogenase (LDH).

• Lactate produced by the LDH reaction is released to the blood stream and
transported to the liver where it is converted to glucose.

• The glucose is then returned to the blood for use by muscle as an energy
source and to replenish glycogen stores. This cycle is termed the Cori cycle.

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Slide 19

SB10 During vigorous muscular activity, epinephrine is released and it stimulates hepatic gluconeogenesis.
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Cori Cycle

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