GLUCONEOGENESIS

Regulation of Glycolysis & Gluconeogenesis

 GLUOCONEOGENESIS

• Is the formation of glucose from non-carbohydrate sources.

• Major site: Liver.
• Minor site: Kidney.

• Liver and kidney are the most important gluconeogenic

tissues; the kidney may contribute up to 40% of total glucose
synthesis in the fasting state and more in starvation.

• Failure of gluconeogenesis is usually fatal.

 SUBSTRATES OF
GLUCONEOGENESIS
• The major substrates are the

1.glucogenic amino acids,

2.lactate,
3.glycerol,
4.and propionate.

• These non-carbohydrate precursors of glucose are

1. first converted into pyruvate
2.or enter the pathway at later intermediates such as oxaloacetate
and dihydroxyacetone phosphate.
 GLUCONEOGENESIS

Glucogenic
amino acids

Lactate, Glycerol (from

pyruvate
Glucose triglycerides)

Propionate Krebs cycle

(ruminants) intermediates
 IMPORTANCE

• Glucose is the only source of energy:

• 1. Nervous system
• 2. Red blood cells

• Glucose is required :
• 1. Adipose tissues: as a source of glycerol.
• 2. Mammary gland: as a source of lactose.
• Gluconeogenesis occurs mainly in liver.
• Gluconeogenesis occurs to a more limited extent in
kidney & small intestine under some conditions.
• Synthesis of glucose from pyruvate utilizes many of the
same enzymes as Glycolysis.

• Three Glycolysis reactions are essentially irreversible.

 Hexokinase (or Glucokinase)
 Phosphofructokinase
 Pyruvate Kinase.
• These steps must be bypassed in Gluconeogenesis.
 MAIN SITES OF GLUCONEOGENESIS:

• The key gluconeogenic enzymes are expressed in the small

intestine, but it is unclear whether or not there is significant
glucose production by the intestine in the fasting state.

• After an overnight fast, glycogenolysis and gluconeogenesis

make approximately equal contributions to blood glucose;

• as glycogen reserves are depleted, so gluconeogenesis becomes

progressively more important.
 REACTIONS OF
GLUCONEOGENESIS
Thermodynamic barriers
• In glycolysis, glucose is converted into pyruvate; in gluconeogenesis,
pyruvate is converted into glucose.
• The formation of glucose does not occur by a simple reversal of
glycolysis, because the overall equilibrium of glycolysis strongly favors
pyruvate formation.
• Instead, glucose is synthesized by a special pathway, gluconeogenesis,
that requires both mitochondrial and cytosolic enzymes.

• So: gluconeogenesis is not a reversal of glycolysis.

• Three reactions in glycolysis catalyzed by

• hexokinase, phosphofructokinase and pyruvate kinase are considered
thermodynamic barriers which prevent simple reversal of glycolysis for
glucose synthesis.
 REACTIONS OF
GLUCONEOGENESIS
In gluconeogenesis, the following new steps bypass these
irreversible reactions of glycolysis:

1. First bypass (Formation of Phosphoenolpyruvate from

pyruvate)

2. Second bypass (Formation of Fructose 6-phosphate from

fructose 1,6-bisphosphate)

3. Third bypass (Formation of Glucose by hydrolysis of glucose

6-phosphate)
 IRREVERSIBLE GLYCOLYTIC
STEPS
BYPASSED
Glycolysis Gluconeogenesis

1. Hexokinase (hexK) by Glucose-6-phosphatase

2. Phosphofructokinase-1 by Fructose 1,6-bisphosphatase

(FBP-1)
(PFK-1)
by Pyruvate Carboxylase &
3. Pyruvate kinase (PyrK) Phosphoenolpyruvate
carboxykinase (PEPCK)
These 3 key enzymes
Gluconeogenesis
as it takes place in
the mammalian
liver.
 SOURCE: GLYCEROL
• Glycerol is released during the hydrolysis of triacylglycerols in
adipose tissue and is delivered by the blood to the liver.

• In the fasting state glycerol released from lipolysis of adipose

tissue triacylglycerol is used solely as a substrate for
gluconeogenesis in the liver and kidneys.

1.Glycerol is phosphorylated by glycerol kinase to glycerol

phosphate,
2.which is oxidized by glycerol phosphate dehydrogenase to
dihydroxyacetone phosphate—an intermediate of glycolysis.

Note: Adipocytes cannot phosphorylate glycerol because they essentially

lack glycerol kinase.
 ENTRY OF GLYCEROL IN TO THE PATHWAY OF
GLUCONEOGENESIS

• Glycerol kinase is absent in adipose tissue, so glycerol released by hydrolysis of

triglycerides can not be utilized for re-esterification, it is a waste product.

• It is carried through circulation to the liver and is used for gluconeogenesis or

glycolysis as the need may be.
 LACTATE
• Lactate is released into the blood by
exercising skeletal muscle, and by cells
that lack mitochondria, such as red
blood cells.

• In the Cori cycle/Lactic acid cycle,

bloodborne glucose is converted by
exercising muscle to lactate, which
diffuses into the blood.

• This lactate is taken up by the liver

and reconverted to glucose, which is
released back into the circulation
 GLUCOSE-ALANINE CYCLE
• This glucose-alanine cycle provides an indirect way of utilizing
muscle glycogen to maintain blood glucose in the fasting state.
• The ATP required for the hepatic synthesis of glucose from pyruvate
is derived from the oxidation of fatty acids.
 AMINO ACIDS
• Amino acids derived from hydrolysis of tissue proteins are the major sources of glucose
during a fast.
• α-Ketoacids, such as α-ketoglutarate, are derived from the metabolism of glucogenic
amino acids.
• These α-ketoacids can enter the TCA cycle and form oxaloacetate (OAA)—a direct
precursor of phosphoenolpyruvate (PEP).

• Note: Acetyl CoA and compounds that give rise only to acetyl CoA (for example,
acetoacetate and amino acids such as lysine and leucine) cannot give rise to a net synthesis
of glucose.

• This is due to the irreversible nature of the pyruvate dehydrogenase reaction, which
converts pyruvate to acetyl CoA. These compounds give rise instead to ketone bodies (and
are therefore termed ketogenic.)
OXALOACETATE
 FIRST BYPASS (FORMATION OF
PHOSPHOENOLPYRUVATE FROM PYRUVATE)

• The first “roadblock” to overcome in the synthesis of glucose

from pyruvate is the irreversible conversion in glycolysis of
PEP to pyruvate by pyruvate kinase.

1.In gluconeogenesis, pyruvate is first carboxylated by

pyruvate carboxylase to OAA,

2.which is then converted to PEP by the action of PEP-

carboxykinase.

• Pyruvate carboxylase is a mitochondrial enzyme, whereas

the other enzymes of gluconeogenesis are cytoplasmic.
 PYRUVATE CARBOXYLASE
 This reaction occurs in the mitochondria of liver and
kidney cells, and has two purposes:

1.to provide an important substrate for gluconeogenesis, &

2.to provide OAA that can replenish the TCA cycle
intermediates that may become depleted, depending on the
synthetic needs of the cell.

 Muscle cells also contain pyruvate carboxylase, but use the

OAA produced only for the latter purpose—they do not
synthesize glucose.
 REACTION CATALYZED BY
PYRUVATE CARBOXYLASE
 Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to
oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme.
 Biotin binds CO2 from bicarbonate as carboxybiotin prior to the addition of the CO 2 to
pyruvate.

• Allosteric regulation:
• Pyruvate carboxylase is allosterically activated by acetyl CoA.
• Elevated levels of acetyl CoA in mitochondria signal a metabolic state in which the
increased synthesis of OAA is required. For example, this occurs during fasting, when
OAA is used for the synthesis of glucose by gluconeogenesis in the liver and kidney.
• Conversely, at low levels of acetyl CoA, pyruvate carboxylase is largely inactive, and
pyruvate is primarily oxidized by the pyruvate dehydrogenase complex to produce
acetyl CoA that can be further oxidized by the TCA cycle.
PYRUVATE CAN GO
“UP” OR “DOWN”
DEPENDING UPON
ENERGY NEEDS
 TRANSPORTATION
OF OXALOACETATE
 Oxaloacetate, the product of the pyruvate carboxylase reaction, is
reduced to malate inside the mitochondrion for transport to the cytosol.
 The reduction is accomplished by an NADH-linked malate
dehydrogenase.
 When malate has been transported across the mitochondrial membrane,
it is reoxidized to oxaloacetate by an NAD+-linked cytosolic malate
dehydrogenase in the cytosol.
 The NADH produced is used in the reduction of 1,3-BPG to
glyceraldehyde 3-phosphate a step common to both glycolysis and
gluconeogenesis
 DECARBOXYLATION
OF OXALOACETATE
A second enzyme, phosphoenolpyruvate carboxykinase, catalyzes the
decarboxylation and phosphorylation of oxaloacetate to
phosphoenolpyruvate using GTP as the phosphate donor.

Biological Significance
• In liver and kidney, the reaction of succinate thiokinase in the citric acid
cycle produces GTP (rather than ATP as in other tissues), and this GTP is
used for the reaction of phosphoenolpyruvate carboxykinase,

• thus providing a link between citric acid cycle activity and

gluconeogenesis.
 Pyruvate Carboxylase PEP Carboxykinase
O O
O C O
O O
C ATP ADP + Pi C O GTP GDP C
C O CH2 C OPO32
CH3 HCO3 C CO2 CH2
O O
pyruvate oxaloacetate PEP

Bypass of Pyruvate Kinase (2 enzymes):

1.Pyruvate Carboxylase (Gluconeogenesis) catalyzes:
pyruvate + HCO3- + ATP  oxaloacetate + ADP + Pi
2. PEP Carboxykinase (Gluconeogenesis) catalyzes:
oxaloacetate + GTP  PEP + GDP + CO2
 O O
P E P C a rb o x y k in a se R e a c tio n
C O
O GTP GDP O O
C O C C
CH2 C O C O P O 32
CO2
C CH2 CH2
O O
o x a lo a c e ta te PEP

PEP Carboxykinase catalyzes GTP-dependent

oxaloacetate  PEP. It is thought to proceed in 2 steps:

 Phosphate transfer from GTP then yields

phosphoenolpyruvate (PEP).
 SECOND BYPASS (FORMATION OF FRUCTOSE 6-
PHOSPHATE FROM FRUCTOSE 1,6-BISPHOSPHATE)
• On formation, phosphoenolpyruvate is metabolized by the
enzymes of glycolysis but in the reverse direction.

• When conditions favor gluconeogenesis, the reverse reactions

will take place until the next irreversible step is reached.

• This step is the hydrolysis of fructose 1,6- bisphosphate to

fructose 6-phosphate and Pi.
 SECOND BYPASS (FORMATION OF
FRUCTOSE 6-PHOSPHATE
FROM FRUCTOSE 1,6-BISPHOSPHATE)
• Fructose 1,6-bisphosphatase catalyzes this exergonic hydrolysis.
• Its presence determines whether a tissue is capable of synthesizing
glucose (or glycogen) not only from pyruvate, but also from triose
phosphates.
• Like its glycolytic counterpart, it is an allosteric enzyme that
participates in the regulation of gluconeogenesis.
 Phosphofructokinase 
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
 Fructose-1,6-biosphosphatase

Phosphofructokinase (Glycolysis) catalyzes:

fructose-6-P + ATP  fructose-1,6-bisP + ADP
Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes:
fructose-1,6-bisP + H2O  fructose-6-P + Pi
 REGULATION BY
FRUCTOSE 2,6-BISPHOSPHATE:

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

inhibited by fructose 2,6 bisphosphate, an allosteric effector
whose concentration is influenced by the level of circulating
glucagon.

• The signals that inhibit (low energy, high fructose 2,6-

bisphosphate) or favor (high energy, low fructose 2,6-
bisphosphate) gluconeogenesis have the opposite effect on
glycolysis, providing reciprocal control of the pathways that
synthesize and oxidize glucose.
 THIRD BYPASS (FORMATION OF
GLUCOSE BY HYDROLYSIS OF GLUCOSE 6-PHOSPHATE)

 The fructose 6-phosphate generated by fructose 1,6-

bisphosphatase is readily converted into glucose 6-phosphate.
 In most tissues, gluconeogenesis ends here.
 Free glucose is not generated; rather, the glucose 6-phosphate
is processed in some other fashion, notably to form glycogen.
 One advantage to ending gluconeogenesis at glucose 6-
phosphate is that, unlike free glucose, the molecule cannot
diffuse out of the cell.
 Glucose-6-phosphatase
6 CH OPO 2 CH2OH
2 3
5 O O
H H H H
H H2O H
4
OH H 1
OH H + Pi
OH OH OH OH
3 2
H OH H OH
glucose-6-phosphate glucose

Hexokinase or Glucokinase (Glycolysis) catalyzes:

glucose + ATP  glucose-6-phosphate + ADP

Glucose-6-Phosphatase (Gluconeogenesis) catalyzes:

glucose-6-phosphate + H2O  glucose + Pi
 THIRD BYPASS (FORMATION OF
GLUCOSE BY HYDROLYSIS OF GLUCOSE 6-PHOSPHATE)
• To keep glucose inside the cell, the generation of free glucose is
controlled in two ways.
1. First, the enzyme responsible for the conversion of glucose 6-phosphate
into glucose, glucose 6-phosphatase, is regulated.

2. Second, the enzyme is present only in tissues whose metabolic duty is to

maintain blood-glucose homeostasis - tissues that release glucose into the
blood.

• These tissues are the liver and to a lesser extent the kidney the enzyme is
absent in muscle and adipose tissue, which therefore, cannot export
glucose into the blood stream.
REACTIONS OF GLUCONEOGENESIS

 In the kidney, muscle and especially

the liver, G6P be shunted toward
glycogen if blood glucose levels are
adequate.
 The G6P produced from
gluconeogenesis can be converted to
glucose-1-phosphate (G1P) by
phosphoglucose mutase (PGM).
 ENERGETICS OF GLUCONEOGENESIS
• Six nucleotide triphosphate molecules are hydrolyzed to synthesize glucose from pyruvate in
gluconeogenesis, whereas only two molecules of ATP are generated in glycolysis in the
conversion of glucose into pyruvate.
• Thus it is not a simple reversal of glycolysis but it is energetically an expensive affair.
• Of the 11 reactions required to convert pyruvate to free glucose, seven are catalyzed by
reversible glycolytic enzymes.
•
• Note: Gluconeogenesis from pyruvate couples the cleavage of six high-energy phosphate bonds
and the oxidation of two NADH with the formation of each molecule of glucose.

• The overall reaction of gluconeogenesis is-

The overall reaction of glycolysis is-

 Glycolysis & Gluconeogenesis

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: 2
1. Glycolysis yields how many ~P ?
6
2. Gluconeogenesis expends how many ~P ?
3. A futile cycle of both pathways would waste how many
~P per cycle ? 4
 GLUCOGENIC AMINO ACIDS
Glucogenic amino acids- Amino acids are derived from the dietary
proteins, tissue proteins or from the breakdown of skeletal muscle
proteins during starvation.
After transamination or deamination, glucogenic amino
acids yield either pyruvate or intermediates of the citric acid cycle.

•Amino acids that are degraded to pyruvate, α-ketoglutarate,

succinyl CoA, fumarate, or oxaloacetate are termed glucogenic
amino acids.

•Amino acids that are degraded to acetyl CoA or Acetoacetyl CoA

are termed ketogenic amino acids because they can give rise to
ketone bodies or fatty acids.
THE ENTRY OF GLUCOGENIC AMINO ACIDS
 ENTRY OF LACTATE IN TO THE
PATHWAY OF GLUCONEOGENESIS
• 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.

Biological Significance:
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.
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.
 Phosphofructokinase 
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
 Fructose-1,6-biosphosphatase

To prevent the waste of a futile cycle, Glycolysis &

Gluconeogenesis are reciprocally regulated.
Local Control includes reciprocal allosteric regulation by
adenine nucleotides.

 Phosphofructokinase (Glycolysis) is inhibited by ATP and

stimulated by AMP.
 Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by
AMP.
The opposite effects of adenine nucleotides on
 Phosphofructokinase (Glycolysis)
 Fructose-1,6-bisphosphatase (Gluconeogenesis)

insures that when cellular ATP is high (AMP would then be

low), glucose is not degraded to make ATP.
When ATP is high it is more useful to the cell to store
glucose as glycogen.
When ATP is low (AMP would then be high), the cell does
not expend energy in synthesizing glucose.
 PFK2/FBPase2 homodimer PDB
2BIF

PFK-2
The allosteric regulator domain
fructose-2,6-bisphosphate is
synthesized & degraded by a
bi-functional enzyme that
FBPase-2
includes 2 catalytic domains: domain
with bound
fructose-6-P
in active site
1. Phosphofructokinase-2 (PFK2) domain catalyzes:
Fructose-6-phosphate + ATP  fructose-2,6-bisphosphate + ADP
2. Fructose-Biophosphatase-2 (FBPase2) domain catalyzes:
Fructose-2,6-bisphosphate + H2O  fructose-6-phosphate + Pi

Bifunctional PFK2/FBPase2 assembles into a homodimer.

 Reciprocal regulation by fructose-2,6-bisphosphate:
 Fructose-2,6-bisphosphate stimulates Glycolysis.
Fructose-2,6-bisphosphate allosterically activates the Glycolysis
enzyme Phosphofructokinase-1.
Gluconeogenesis is stimulated by a decrease of in F2,6bisphosphate.
Fructose-2,6-bisphosphate also activates transcription of the
gene for Glucokinase, the liver variant of Hexokinase that
phosphorylates glucose to glucose-6-phosphate, the input to
Glycolysis.
 Fructose-2,6-bisphosphate allosterically inhibits the
gluconeogenesis enzyme Fructose-1,6-bisphosphatase.
 Glycogen Pyruvate

X Gluconeogenesis

Glucose-1-P Glucose-6-P Glucose + Pi

Glucose-6-Pase
X

Glycolysis
Pathway

Summary of effects of glucagon-cAMP cascade in liver:

 Gluconeogenesis is stimulated.
 Glycolysis is inhibited.
 Glycogen breakdown is stimulated.
 Glycogen synthesis is inhibited.
 Free glucose is formed for release to the blood.
• 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.

Liver Blood Muscle

Glucose Glucose
2 NAD+ 2 NAD+
2 NADH 2 NADH
6 ~P 2 ~P

2 Pyruvate 2 Pyruvate
2 NADH 2 NADH
2 NAD+ 2 NAD+
2 Lactate 2 Lactate
 Liver Blood Cancer Cell

Glucose Glucose
2 NAD+ 2 NAD+
2 NADH 2 NADH
6 ~P 2 ~P

2 Pyruvate 2 Pyruvate
2 NADH 2 NADH
2 NAD+ 2 NAD+
2 Lactate 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.
 CORI CYCLE: LACTATE
SALVAGE

6 ATP
GN
2

RBCs

2 ATP
GL
 GLUCONEOGENESIS
FROM STORED FATS
Triacylglycerols
• In animals Fatty acids CH2OH
Glycerol CHOH
Glycerol kinase
CH2OH
Glycerol-P
Glycerol-P DH
Dihydroxyacetone-P
Triose-P isomerase
Glyceraldehye-3-P
Gluconeogenesis
Glucose
 GLUCONEOGENESIS FROM
PROPIONATE
Cellulose, etc...
Protozoa, bacteria ferment
• In ruminants Propionate
Propionyl-CoA carboxylase
Methylmalonyl-CoA
B12
Succinyl-CoA
Krebs cycle
Oxaloacetate

PEP

Glucose Galactose Lactose

 “IT IS INCORRECT TO SAY THAT
FATS CAN NOT BE CONVERTED TO GLUCOSE”
• Triglycerides (fats) on hydrolysis yield fatty acids and glycerol.
• Even chain fatty acids are not the glucogenic precursors,
• Oxidation of these fatty acids yields enormous amounts of energy,
however, the carbons of the fatty acids cannot be utilized for net
synthesis of glucose.
• The two carbon unit of acetyl-CoA derived from β-oxidation of fatty
acids can be incorporated into the TCA cycle, however, during the TCA
cycle two carbons are lost as CO2.
• Moreover the formation of acetyl CoA from pyruvate is an irreversible
step, thus acetyl CoA can not be converted back into glucose.
• Odd chain fatty acids on oxidation produce Propionyl co A which is a
substrate for gluconeogenesis through formation of succinyl co A.
• Glycerol component of fats can also be utilized for the formation of
glucose through formation of Dihydroxyacetone phosphate.
• Hence therefore except for even chain fatty acids, the other fat
components are glucogenic, so the above given statement that “It is
incorrect to say that fats can not be converted to glucose”, is a justified
statement.