Chapter 17

Gluconeogenesis

Why Gluconeogenesis?
Gluconeogenesis is the synthesis of glucose from
noncarbohydrate precursors (pyruvate, amino acids, glycerol).
Gluconeogenesis is especially important during fasting or
starvation, as glucose is the primary fuel for the brain and the
only fuel for red blood cells (no mitochondria), and the reserve of
glucose in body is used up in ~ 1 day without eating food. (During
intense exercise, gluconeogenesis also takes place.)
The major site of gluconeogenesis is the liver. Small amount of
gluconeogenesis can also occur in the kidney.
Gluconeogensis in liver and kidney maintain the glucose level in
blood, from which it can be extracted by the brain and muscle to
meet their needs.

 The gluconeogenic pathway converts pyruvate into glucose.

Other noncarbohydrate precursors, including lactate, amino acids
and glycerol, are converted to pyruvate or later intermediates.
A. Lactate (muscle-derived) can be converted to pyruvate in the
liver (reverse of the lactic acid fermentation).

 B. The carbon skeletons of most amino acids (almost all,

only exceptions are lysine and leucine) can be converted
into glucogenic intermediates (pyruvate, oxaloacetate) that
enter gluconeogenesis.
C. Glycerol (end product of hydrolysis of triacylglycerols),
can be converted into dihydroxyacetone phosphate
(DHAP), which can enter either gluconeogenesis or
glycolysis.

Most enzymes (7 enzymes) that can catalyze the reversible reactions

are common in glycolysis and gluconeogenesis.
However, the three irreversible steps in glycolysis are too
energetically favorable in one direction and must be bypassed in
gluconeogenesis due to large free energy cost in the reverse
direction.

phosphoglucose
isomerase
Phosphofructose
kinase

Aldolase

Glyceraldehyde 3phosphate dehydrogenase

Phosphoglycerate
kinase

Phosphoglycerate
mutase
Enolase

Triose
phosphate
isomerase

Stage 1 of Gluconeogenesis (7 reactions)

Two enzymes: Pyruvate
carboxylase (in
mitochondria) and PEP
carboxykinase (in
cytoplasm) are used to
reverse the action of
pyruvate kinase.
These occur at a cost of 1
ATP and 1 GTP per pyruvate
that involves a carboxylation
and a decarboxylationcoupled phosphorylation.
The other reactions in this
stage use the same enzymes
in glycolysis.

2X

Stage 2 of Gluconeogenesis (4 reactions)

Fructose 1,6bisphosphatase (that
catalyzes hydrolysis of
a phosphoryl group) is
used to reverse PFK.

Glucose-6-phosphatase is used to
reverse hexokinase.
The hydrolysis of phosphoryl
group in these two steps are
energetically favorable.
To form 1 glucose from 2
pyruvate requires total 6 NTPs.

The formation of phosphoenolpyruvate from pyruvate requires two

enzymes and the reactions occur in mitochondria and cytoplasm,
separately. The sum of the two reactions is:

mitochondria

Phosphoenolpyruvate
carboxykinase

cytoplasm

Pyruvate Carboxylase
Pyruvate carboxylase requires the vitamin biotin (VB7) as a cofactor.

Biotin, a CO2 carrier

The formation of oxaloacetate (OA) by pyruvate carboxylase occurs in

three steps (involves four substrates, sequential mechanism).
Step 1: Activation of bicarbonate by ATP (phosphorylation)

Step 2 : Activated CO2 group is transferred to the biotin-enzyme

complex covalently.

Carboxylated biotin-enzyme
Step 3 : Carboxylate group is transferred to the substrate pyruvate.

Swing Arm of the Biotin Coenzyme

The long flexible linker between biotin and the enzyme
(provided by 5C side chain on biotin and 4C lysine)
enables the carboxy-biotin to rotate from one active site
(ATP-biocarbonate site) to the other (pyruvate site)
sequentially.

Biotin

Acetyl CoA (entry point of citric acid cycle) allosterically activate

the pyruvate carboxylase (entry point of gluconeogenesis, the
product oxaloacetate is also in the entry point of citrate acid cycle).
This important control mechanism balances the two substrates for
citrate synthase.
Pyruvate
pyruvate carboxylase

citric acid cycle

Transport of oxaloacetate from mitochondria

matrix to cytoplasm
Pyruvate to oxaloacetate conversion
occurs in mitochondrion matrix.
In order to be translocated to the
cytoplasm, oxaloacetate is reduced
by NADH to malate by malate
Malate
dehydrogenase.
dehydrogenase
Malate is able to be transported from
mitochondrion matrix into the
cytoplasm (by malate transporter).
Malate is then oxidized back to
oxaloacetate by NAD+ and form
Malate
cytoplasmic NADH.
dehydrogenase

Decarboxylation and phosphorylation of oxaloacetate to

form phosphoenolpyruvate
Phosphoenolpyruvate
carboxykinase

Hydrolysis of GTP does not provide enough free energy to

phosphorylate oxaloacetate. As a result, decarboxylation, which
releases energy, is needed to drive the reaction forward (the reason
for the carboxylation in the first step at the cost of an ATP)
Two enzymes (pyruvate carboxylase and phosphenolpyruvate
carboxykinase) catalyze the phosphorylation of pyruvate at the cost
of two NTPs. The location of reaction also switch from mitochondrion
matrix to cytoplasm though a malate transporter (redox reaction).

 Phosphoenolpyruvate (PEP) is metabolized by the same series of

enzymes of glycolysis in the reverse direction in cytoplasm
(enolase, phosphoglycerate mutase (PGM), phosphoglycerate
kinase (PGK), GAPDH, TIM, aldolase, in condition that favors
gluconeogenesis) until the next irreversible step: the hydrolysis
of fructose 1,6-bisphosphate.
The enzyme catalyzing this reaction is fructose 1,6bisphosphatase, an allosteric enzyme that participate in
regulation.

G6P is Converted to Glucose in ER

F6P->G6P isomerization is easy and reversible. In most tissues,
gluconeogenesis stops here and G6P is used to form glycogen.
The generation of free glucose occurs essentially only in liver,
and is the final irreversible step in gluconeogenesis.
G6P is transported into the lumen of the endoplasmic reticulum.
Glucose 6-phosphatase, an integral membrane protein on the
inner surface of the endoplasmic reticulum, catalyzes the
formation of glucose from G6P.
Glucose is then transported to cytoplasm and to blood.

Net reaction of gluconeogenesis:

Compare with direct reverse of glycolysis

2

Hydrolysis of 4 more NTPs makes the gluconeogenesis reactions

energetically favorable. (but costly)

Gluconeogenesis
4 ATP, 2 GTP expenditure

 Gluconeogenesis and glycolysis are both energetically favorable

pathways (both have overall large negative free energy changes).
Therefore, there is no thermodynamic barrier to prevent them from
taking place simultaneously.
The higher energy cost of gluconeogenesis (6 NTP vs. 2 ATP) means
simultaneous activation of both pathways is not energy efficient.
Gluconeogenesis and glycolysis are regulated so that within a cell,
one pathway is relatively inactive while the other is highly active.
This is known as reciprocal regulation.
Glycolysis will predominate when glucose is abundant, and
gluconeogenesis will be more active when glucose is scarce.

When ATP is required (AMP and ADP are

high) and glucose is abundant (F-2,6-BP,
F-1,6-BP are high), glycolysis
predominates.
When the energy charge is high and
biosynthetic intermediates are abundant
(ATP, citrate, alanine, acetyl CoA are
high), gluconeogenesis will take over.
The inter-conversion of F-1,6-BP and F-6-P
by PFK or Fructose 1,6-bisphosphatase is
a key regulatory site.
Inter-conversion of PEP and pyruvate is
also regulated.
F-2,6-BP and AMP stimulates PFK and inhibits
F-1,6-BPase. Citrate acts oppositely.
PFK is also inhibited by ATP and low pH.

F-1,6-BP stimulates PK.

ADP inhibits PC and PEPCK.
ATP and alanine inhibit PK.
Acetyl-CoA stimulate PC.

Bifunctional PFK2/FBPase2
The key regulator molecule of
glucose metabolism in liver is F-2,6BP, which stimulates PFK and inhibits
FBPase.
The kinase that synthesizes F-2,6-BP
and the phosphatase that hydrolyzes
it are located on the same
polypeptide chain as two linked
domains (PFK2/FBPase2), thus is
called a bifunctional enzyme.

Kinase: F6P -> F2,6BP

Phosphatase: F2,6BP -> F6P

Regulation of PFK2/FBPase2 by Glucagon

Glucagon, a peptide hormone, functions to raise glucose level, opposite to
insulin. Together they maintain a relative constant blood glucose level.
When blood glucose is low, glucagon level is high. Glucagon signaling pathway
leads to phosphorylation of the bifunctional enzyme (by protein kinase A),
which inhibits the kinase (no F-2,6BP, low PFK activity, slows down glycolysis),
and stimulates the phosphatase (favors gluconeogenesis).
When blood glucose is high, glucagon level is low. This leads to
dephosphorylation of the bifunctional enzyme, that activate the kinase and
produces more F-2,6BP, thus stimulating PFK to turn on glycolysis.

Substrate cycle: combinations of two

pathways in which the final products from
one is exactly the initial reactants of the
other, and vice versa. Also referred to as
futile cycles.
Despite reciprocal control, one pathway is
never completely shut down. Why dont
the body prevent futile cycles?
One hypothesis: substrate cycles can
amplify a metabolic signal to increase the
substrate flux down a metabolic pathway
without changing the rates of opposing
reactions too much.

The Cori Cycle Converting Lactate back to Glucose

Skeletal muscle (as well as red blood cell) and liver display interorgan cooperation in a series of reactions called the Cori cycle.

Lactate produced by skeletal muscle and red blood cells is

released into the blood.

Liver removes most of the lactate in blood and either

converts it back into glucose (via gluconeogenesis), which can be
released into the blood available to other tissues, or use the
converted pyruvate for energy generation further down the citric
acid cycle.

The Cori Cycle Converting Lactate back to Glucose

Anaerobic metabolism

Both processes are active in the body but isolated in different organs.
Blood transport the precursors and products around to where these
are need. Membrane transporters allow them to be transported in and
out of the cells.

Alanine, like lactate, is a major precursor of glucose in liver.

Alanine is generated in muscle cells when the carbon skeleton of
some amino acids are used to generate energy, the amino group
goes to pyruvate to form alanine, which is released into blood.
Liver takes up the alanine and converts it back to pyruvate and
then glucose that can be used by other tissues.

lactate

alanine

pyruvate

or Alanine

or Alanine

Summary
1. Glucose can be synthesized from noncarbohydrate
precursors (pyruvate, lactate, amino acids, glycerol).
Gluconeogenesis requires four new reactions (enzymes) to
bypass the three irreversible reactions in glycolysis.
1. Gluconeogenesis and glycolysis are reciprocally regulated,
so both are not maximally active at the same time. Key
regulation points are the phosphofructokinase and fructose
1,6-bisphosphatase by F-2,6-BP. Pyruvate kinase and
pyruvate carboxylase are also regulated by other effectors
(ATP, ADP, AMP, alanine, F-1,6-BP, acetyl CoA) so they
are not active at the same time.
2. Precursors (lactate, alanine) produced by muscle are used
to synthesize glucose in liver that can be used by other
organs.

The following sequence is a part of the sequence of reactions in gluconeogenesis.

Match the capital letters representing the reaction in the gluconeogenic pathway
with parts a, b, c, etc.
(a) takes place in mitochondria.
(b) takes place in the cytoplasm.
(c) produces CO2.
D
A
(d) consumes CO2.
B
(e) requires NADH.
C
(f) produces NADH.
A
(g) requires ATP.
D
(h) requires GTP.
none
(i) requires thiamine.
A
(j) requires biotin.
(k) is regulated by acetyl CoA. A

A, B
C, D

Counting high-energy compounds:

How many NTP molecules are required to synthesize glucose
from each of the following compounds?
(a) Glucose 6-phosphate

none

(b) Fructose 1,6-bisphosphate

(c) Two molecules of oxaloacetate

none
2 ATP + 2 GTP

(d) Two molecules of dihydroxyacetone phosphate none

(e) Pyruvate

6 NTP

(f) Lactate

6 NTP

(g) 3-phosphoglycerate

2 ATP

Match up. Indicate which of the conditions listed in the righthand column increase the activity of the glycolytic and
gluconeogenic pathways.
2 3
1

9
7

Gluconeogenesis takes place in liver during intense exercise,

which seems counterintuitive. Why would an organism
synthesize glucose and, at the same time, use glucose to
generate energy?
Compare the roles of lactate dehydrogenase in gluconeogenesis
and in lactic acid fermentation.