Transcript Gluconeogenesis Reading:  Harper’s Biochemistry Chapter 21  Lehninger Principles of Biochemistry 3rd Ed.

Gluconeogenesis
Reading:
 Harper’s Biochemistry Chapter 21
 Lehninger Principles of Biochemistry
3rd Ed. pp. 723-733
OBJECTIVES
1. To understand how blood glucose levels are
regulated by hormones, especially epinephrine,
glucagon, and insulin.
2. To examine metabolic consequences of loss of
glucose homeostasis.
3. To understand how glucose is synthesized from
other substrates, and which substrates can be
used for this purpose.
4. To understand how glycolysis and
gluconeogenesis are coordinately regulated so as
to avoid futile cycles in the cell.



Gluconeogenesis is the term used to include all
mechanisms and pathways responsible for
converting non-carbohydrates to glucose or
glycogen.
The major substrates are the glucogenic amino
acids; lactate; glycerol; and propionate.
Gluconeogenesis occurs in the liver and kidney, the
only organs with a full complement of the necessary
enzymes.
Biomedical Importance





The biosynthesis of glucose is an absolute necessity of all
mammals, because the brain and nervous system, as well as
erythrocytes, testes, renal medulla, and embryonic tissue,
require glucose from the blood as their sole or major fuel
source.
The human brain alone requires 120 g of glucose each day.
Below a critical blood glucose concentration (normal = 65110 mg/dL or 3.6-6 mM), brain dysfunction can occur which
can lead to coma and death.
Even when fat may be supplying most of the caloric
requirements of an organism, there is always a certain basal
requirement for glucose e.g. in skeletal muscle under
anaerobic conditions.
Glucose is precursor of lactose in the mammary gland.
Gluconeogenic mechanisms are used to clear lactate (from
muscle and erythrocytes) and glycerol (adipose tissue) from
blood.
Gluconeogenesis vs. Glycolysis




Thermodynamic barriers prevent a simple reversal of
glycolysis in conversion of pyruvate to glucose.
7 of 10 reactions of gluconeogenesis are the reverse of
glycolytic reactions.
Three reactions of glycolysis are essentially irreversible in
vivo and cannot be used in gluconeogenesis
- the conversion of glucose to glucose 6-phosphate
- the phosphorylation of fructose 6-phosphate to fructose
1,6-bisphosphate by phosphofructokinase-1
- the conversion of phosphoenolpyruvate to pyruvate by
pyruvate kinase.
In cells, these three reactions are characterized by a large
negative G, whereas other glycolytic reactions have a G
near zero and can be reversed in vivo during
gluconeogenesis.
BYPASS REACTIONS
1. Conversion of pyruvate to
phosphoenolpyruvate




Pyruvate is first transported into the mitochondria from the
cytosol, or generated from alanine by transamination
within mitochondria. Pyruvate is converted to oxaloacetate
by pyruvate carboxylase which requires biotin:
Pyruvate + HCO3- + ATPoxaloacetate + ADP + Pi
Pyruvate carboxylase requires acetyl-CoA as a positive
effector, and biotin acts as a carrier of activated HCO3The oxaloacetate formed is reduced to malate by
mitochondrial malate dehydrogenase:
oxaloacetate + NADH + H+
L-malate + NAD+
Malate leaves the mitochondrion and is re-oxidized to
oxaloacetate, with production of cytosolic NADH
Malate + NAD+ oxaloacetate + NADH + H+


The oxaloacetate is then converted to
phosphoenolpyruvate (PEP) by phosphoenolpyruvate
carboxykinase, requiring Mg2+ and GTP:
oxaloacetate + GTP PEP + CO2 + GDP
Overall equation for this set of bypass reactions:
Pyruvate + ATP + GTP + HCO3- 
PEP + ADP + GDP + Pi + CO2

Two high-energy phosphate equivalents must be
expended to phosphorylate one molecule of pyruvate
to PEP
BYPASS REACTIONS
2. Conversion of fructose 1,6-bisphosphate
to fructose 6-phosphate

This is catalyzed by Mg2+ -dependent fructose
1,6-bisphosphate which hydrolyzes the C-1
phosphate
fructose 1,6-bisphosphate + H2O
fructose 6-phosphate
G´° = -16.3 kJ/mol
BYPASS REACTIONS
3. Conversion of glucose 6-phosphate to
free glucose

This is catalyzed by glucose 6-phosphatase,
a Mg2+ -activated enzyme, and involves a
simple hydrolysis of a phosphate ester:
glucose 6-phosphate + H2O glucose + Pi

The enzyme is found in hepatocytes and
renal cells, but not in muscle or brain, and is
located on the lumenal side of the ER
membrane
Citric acid cycle intermediates and
many amino acids are glucogenic



The biosynthetic pathway of gluconeogenesis allows
the net synthesis of glucose from pyruvate and also
from the citric acid cycle intermediates citrate,
isocitrate, -ketoglutarate, succinyl-CoA, succinate,
fumarate, and malate, all of which can undergo
oxidation to oxaloacetate.
Some or all of the carbon atoms of many of the amino
acids derived from protein are ultimately converted to
pyruvate or to intermediates in the citric acid cycle.
Such amino acids are said to be glucogenic.
Alanine and glutamine are particularly important
because they are the primary molecules that transport
amino groups from extrahepatic tissue to the liver.
Futile Cycles in carbohydrate
metabolism consume ATP

Simultaneous operation of parallel steps in glycolytic and
gluconeogenic pathways would be wasteful, e.g. reactions
catalyzed by phosphofructokinase-1 and fructose 1,6bishphosphatase ATP + fructose 6-phosphate 
ADP + fructose 1,6-bisphosphate
fructose 1,6-bisphosphate + H2O 
fructose 6-phosphate + Pi
NET REACTION: ATP + H2O  ADP + Pi + heat



This is called a futile cycle and could in principle occur with
other sets of reactions.
Futile cycling is prevented by reciprocal regulatory mechanisms
Futile cycling can be used to generate heat (e.g. bumble bee use
above reaction to warm muscles in cold weather)
Reciprocal regulation of
gluconeogenesis and glycolysis


1st control point - fate of
pyruvate
Two alternative fates for
pyruvate. Pyruvate can be
converted to glucose and
glycogen via
gluconeogenesis or
oxidized to acetyl-CoA for
energy production. The
first enzyme in each path
is regulated allosterically;
acetyl-CoA stimulates
pyruvate carboxylase and
inhibits the pyruvate
dehydrogenase complex

2nd control point - fructose 1,6-bisphosphate and
phosphofructokinase
Glucose
Fructose 6-phosphate
AMP
citrate
ATP
AMP, ADP
Fructose 1,6-bisphosphate
Citric Acid Cycle
ATP

Fructose 2,6-bisphosphate plays a unique role in the
regulation of glycolysis and gluconeogensis in liver

Fructose 2,6-bisphosphate is an allosteric effector for
the enzymes phosphofructokinase-1 and fructose 1,6bisphosphatase.
Fructose 2,6-bisphosphate activates PFK-1 and
stimulates glycolysis in liver
At the same time, Fructose 2,6-bisphosphate inhibits
Fructose 1,6-bisphosphatase, thereby slowing
gluconeogenesis
Fructose 2,6-bisphosphate is not an intermediate in
these pathways, but a regulator




Fructose 2,6-bisphosphate activates PFK-1 and
inhibits FBPase-1, stimulating glycolysis and
inhibiting gluconeogenesis


Fructose 2,6-bisphosphate levels are regulated by rates of
synthesis by PFK-2 and breakdown by FBPase-2
Regulation of fructose 2,6-bisphosphate level, (a) The
cellular concentration of the regulator fructose 2,6bisphosphate is determined by the rates of its synthesis by
PFK-2 and breakdown by FBPase-2. (b) Both of these
enzymes are part of the same polypeptide chain, and both
are regulated, in a reciprocal fashion, by glucagon. Here
and elsewhere, arrows are used to indicate increasing and
decreasing levels of metabolites.