Gluconeogenesis,,,,,,,,,,,,,,,,,,,,,,,,,

Gluconeogenesis
Dr. Joanne Sadier

ILOs
 List gluconeogenic precursor
 List the enzymes and intermediates involved in gluconeogenesis’
 Recognize the reversible and regulated steps of gluconeogenesis
 Discuss how blood glucose level is regulated and which hormones are involved
 Describe the mechanism of action of each of the Insulin, glucagon and epinepherin
hormones
 Discuss regulation of gluconeogenesis

Introduction
 The pathway for gluconeogenesis shares some steps with glycolysis, the pathway for
glucose degradation, but four reactions specific to the gluconeogenic pathway are not
found in the degradation pathway.
 These reactions replace the metabolically irreversible reactions of glycolysis. These
opposing sets of reactions are an example of separate, regulated pathways for
synthesis and degradation

 In addition to fueling the production of ATP
(via glycolysis and the citric acid cycle),
glucose is also a precursor of the ribose and
deoxyribose moieties of nucleotides and
deoxynucleotides.
 The pentose phosphate pathway is
responsible for the synthesis of ribose as well
as the production of reducing equivalents in
the form of NADPH.
Glucose availability is controlled by regulating the
uptake and synthesis of glucose and related molecules
and by regulating the synthesis and degradation of
storage polysaccharides composed of glucose
residues. Glucose is stored as glycogen in bacteria and
animals and as starch in plants

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• Glucose is stored as starch and
glycogen
• Glycogen is stored in cytosolic
granules in muscle and liver cells of
vertebrates
• Glycogenolysis - degradation of
glycogen
• Glycogen breakdown yields G1P
which can be converted to G6P for
metabolism via glycolysis and the
citric acid cycle
Glycogen Degradation
Muscle glycogen appears in electron micrographs as cytosolic
granules with a diameter of 10 to 40 nm, about the size of
ribosomes

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Cleavage of a glucose residue from the
nonreducing end of glycogen
Glycogen Phosphorylase

Glycogen phosphorylase
 Glycogen phosphorylase is
responsible for the breakdown of
glycogen to produce glucose 1-
phosphate.
 In muscle cells, glucose 1-phosphate
is converted to glucose 6-phosphate
that is used in glycolysis to produce
ATP.
 In liver cells, glucose 6-phosphate is
hydrolyzed to free glucose that is
secreted into the bloodstream where it
can be taken up by other tissues

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Metabolism of Glucose 1-Phosphate (G1P)
• Phosphoglucomutase catalyzes the conversion
of G1P to glucose 6-phosphate (G6P)

Glycogen synthesis
 Glycogen synthesis is a polymerization
reaction where glucose units are added
one at a time to a growing polysaccharide
chain.
 This reaction is catalyzed by glucogen
synthase.
 Many polymerization reactions are
processive—the enzyme remains bound
to the end of the growing chain and
addition reactions are very rapid
 The glycogen synthase reaction is
distributive—the enzyme releases the
growing glycogen chain after each
reaction.

10
Glycogen Synthesis
• Synthesis and degradation of glycogen
require separate enzymatic steps
• Cellular glucose converted to G6P by
hexokinase
• Three separate enzymatic steps are
required to incorporate one G6P into
glycogen
• Glycogen synthase is the major
regulatory step

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Glycogen synthase adds glucose to
the nonreducing end of glycogen

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Regulation of Glycogen
Metabolism
• Muscle glycogen is fuel for muscle contraction
• Liver glycogen is mostly converted to glucose for
bloodstream transport to other tissues
• Both mobilization and synthesis of glycogen are
regulated by hormones
• Insulin, glucagon and epinephrine regulate
mammalian glycogen metabolism

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Hormones Regulate Glycogen
Metabolism
• Insulin is produced by b-cells of
the pancreas (high levels are
associated with the fed state)
• Insulin increases rate of glucose
transport into muscle, adipose
tissue via GLUT 4 transporter
• Insulin stimulates glycogen
synthesis in the liver

 Effect of insulin on glycogen
metabolism.
 Insulin simulates the phosphatase
activity of phosphoprotein
phosphatase-1, leading to inactivation
of glycogen phosphorylase and
activation of glycogen synthase.

Gluconeogenesis
 large mammals that have not eaten for 16 to
24 hours have depleted their liver glycogen
reserves and need to synthesize glucose to
stay alive
 Some mammalian tissues, primarily liver
and kidney, can synthesize glucose from
simple precursors such as lactate and
alanine.
Note that many of the intermediates and enzymes are identical.
All seven of the near-equilibrium reactions of glycolysis proceed in
the reverse direction during gluconeogenesis

Inter-talk between muscle and liver
 Under fasting conditions,
gluconeogenesis supplies almost all of
the body’s glucose.
 When exercising under anaerobic
conditions, muscle converts glucose to
pyruvate and lactate, which travel to
the liver and are converted to glucose

2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H2O + 2 H+
Glucose + 2 NAD+
+ 4 ADP + 2 GDP + 6 Pi
Gluconeogenesis
Recall that glycolysis consumes two ATP molecules and generates four, for a net yield
of two ATP equivalents and two molecules of NADH.
Contrast this with the synthesis of one molecule of glucose by gluconeogenesis
consuming a
total of six ATP equivalents and two molecules of NADH.
As expected, the biosynthesis of glucose requires energy and its degradation releases
energy.

Pyruvate Carboxylase
 two enzymes required for
synthesis of phosphoenolpyruvate.
 The two steps involve a
carboxylation followed by
decarboxylation
 Pyruvate carboxylase catalyzes a
metabolically irreversible reaction
—it can be allosterically activated
by acetyl CoA.
Oxaloacetate can enter the citric acid
cycle or serve as a precursor for
glucose biosynthesis.

 Pyruvate carboxylase catalyzes a
metabolically irreversible reaction—it
can be allosterically activated by
acetyl CoA.
 This is the only regulatory mechanism
known for the enzyme.
 High amount of acetyl CoA indicates
that it is not being efficiently
metabolized by the citric acid cycle.
 Under these conditions, pyruvate
carboxylase is stimulated in order to
direct pyruvate to oxaloacetate
Pyruvate Carboxylase

Phosphoenolpyruvate
Carboxykinase (PEPCK)
 The enzyme found in bacteria, protists,
fungi, and plants uses ATP while The
animal version uses GTP.
 In most species, the enzyme displays
no allosteric kinetic properties and has
no known physiological modulators. Its
activity is most often affected by
controls at the level of transcription of
its gene

Protein regulation at the
transcription level
In the fasting state
Prolonged release of glucagon from the
pancreas leads to continued elevation of
intracellular cAMP, that triggers increased
transcription of the PEPCK gene in the liver
and increased synthesis of PEPCK.
After several hours, the amount of PEPCK
rises and the rate of gluconeogenesis
increases
In the fed state
Insulin, abundant in the fed state, acts in
opposition to glucagon at the level of the
gene reducing the rate of synthesis of
PEPCK

Fructose 1,6-bisphosphatase
 The reactions of gluconeogenesis
between phosphoenolpyruvate and
fructose 1,6- bisphosphate are simply the
reverse of the near-equilibrium reactions
of glycolysis.
 The next reaction in the glycolysis
pathway—catalyzed by
phosphofructokinase-1—is metabolically
irreversible

Regulation of
Phosphofructokinase-1 PFK-1
 ATP is a substrate and an allosteric inhibitor of
PFK-1
 The bacterial enzyme is activated by ADP but
in mammals AMP is the allosteric activator of
PFK-1. AMP acts by relieving the inhibition
caused by ATP.
 Citrate, an intermediate of the citric acid cycle,
is another physiologically important inhibitor of
mammalian PFK-1.
 An elevated concentration of citrate indicates
that the citric acid cycle is blocked and further
production of pyruvate would be pointless.

fructose 1,6-bisphosphate
regulation
 The two enzymes that catalyze the
interconversion of fructose 6-
phosphate and fructose 1,6-
bisphosphate are reciprocally
controlled by the concentration of
fructose 2,6-bisphosphate

Glucose 6-phosphatase
 Although we present glucose as the
final product of gluconeogenesis, this
is not true in all species.
 In most cases, the biosynthetic
pathway ends with glucose 6-
phosphate.
 This product is an activated form of
glucose. It becomes the substrate for
additional carbohydrate pathways
leading to synthesis of glycogen,
starch and sucrose, pentose sugars,
and other hexoses.

 In these cells, glucose 6-phosphatase is bound to the
endoplasmic reticulum with its active site in the lumen.

 The enzyme is part of a complex that includes a glucose
6-phosphate transporter (G6PT) and a phosphate
transporter.
 G6PT moves glucose 6-phosphate from the cytosol to
the interior of the ER where it is hydrolyzed to glucose
and inorganic phosphate.
 Phosphate is returned to the cytosol and glucose is
transported to the cell surface (and the bloodstream) via
the secretory pathway.
Glucose is made in the cells of the liver, kidneys, and
small intestine and exported to the bloodstream

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• A metabolically irreversible hydrolysis reaction
• GLUT7 transporter conveys G6P from cytosol to
enzyme on the endoplasmic reticulum membrane
(liver, kidney, pancreas, small intestine)
• Glucose is exported from the ER to bloodstream

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• (continued next slide)
Comparison of
gluconeogenesis
and glycolysis

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Precursors for Gluconeogenesis
• Any metabolite that can be converted to pyruvate
or oxaloacetate can be a glucose precursor
• Major gluconeogenic precursors in mammals:
(1) Lactate
(2) Most amino acids (especially alanine),
(3) Glycerol (from triacylglycerol hydrolysis)

Precursors for Gluconeogenesis
 The major gluconeogenic precursors in
mammals are lactate and most amino
acids, especially alanine.
 Glycerol, which is produced from the
hydrolysis of triacylglycerols, is also a
substrate for gluconeogenesis. Glycerol
enters the pathway after conversion to
dihydroxyacetone phosphate.

 We will talk in detail about glycerol 3 P
dehydrogenase complex in future
chapter
Gluconeogenesis from Glycerol

Lactate
• Glycolysis generates large
amounts of lactate in active muscle
• Red blood cells steadily produce
lactate
• Liver lactate dehydrogenase
converts lactate to pyruvate (a
substrate for gluconeogenesis)
• Glucose produced by liver is
delivered to peripheral tissues via
the bloodstream
• The interaction of glycolysis and gluconeogenesis-
the Cori cycle

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Amino Acids
• Carbon skeletons of most amino acids are catabolized to pyruvate or
citric acid cycle intermediates
• The glucose-alanine cycle:
(1) Transamination of pyruvate yields alanine which travels to the liver
•
(2) Transamination of alanine in the liver yields pyruvate for
gluconeogenesis
• (3) Glucose is released to the bloodstream

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Subcellular Locations of
Gluconeogenic Enzymes
• Gluconeogenesis enzymes are cytosolic except:
(1) Glucose 6-phosphatase (endoplasmic reticulum)
(2) Pyruvate carboxylase (mitochondria)
(3) PEPCK (cytosol and/or mitochondria)

Gluconeogenesis,,,,,,,,,,,,,,,,,,,,,,,,,

Gluconeogenesis,,,,,,,,,,,,,,,,,,,,,,,,,

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