You are on page 1of 12

[BIOCHEM] 2.

7
Gluconeogenesis
[BIOCHEM] 2.7 Gluconeogenesis
Dr.
Magat
Dr. M. Magat

August 8, 2013

Ferrer, A (09054793305), Fernandez J, Fernandez M, Ferranco, Ferrer C, Figueroa, Flores

OUTLINE

I.

I. Overview of Gluconeogenesis
II. Important cycles between tissues that involve
Gluconeogenesis
A. Cori cycle (Glucose Lactate cycle)
B. Alanine cycle (Glucose Alanine cycle)
III. Pathways of Glucose Synthesis
A. Gluconeogenesis from Amino Acids
B. Gluconeogenesis from Triacylglycerol
C. Gluconeogenesis from Other Sugars
IV. Metabolic Steps of Gluconeogenesis
V. Gluconeogenic Enzymes
VI. Regulation
A. Expenditure of ATP
B. Regulation of gluconeogenesis
C. Hormonal control
D. Fates of lactate formed during muscle
contraction
E. PEPCK regulation in promoter region
VII. Summary
VIII. Appendix
IX. Study Guide
OBJECTIVES
At the end of the lecture, the student should be able to:
1. Appreciate that glucose can be synthesized from lactate,
pyruvate, amino acids, triacylglycerides, and fructose
2. Understand that gluconeogenesis is mostly the reverse of
glycolysis with a few bypassed reactions
3 Discuss the metabolic steps of gluconeogenesis
4. Discuss the regulation of gluconeogenesis
References:
Devlin, Thomas M. Textbook of Biochemistry with Clinical
Correlations. 7th ed.: Wiley, 2010.
Murray, Robert K. Harper's Illustrated Biochemistry. 29th
ed.: McGraw-Hill, 2012. Print.
Nelson, David L. Lehninger's Principles of Biochemistry. 5th
ed. 2009.
Stryer, Lubert. Biochemistry. 6th ed. 2006.
Marks, Allan D. Mark's Basic Medical Biochemistry a Clinical
Approach. 2nd ed., 2005.
Grisham, Charles M. Biochemistry. 4th ed. 2010.
Voet, Donald J. Principles of Biochemistry.: Wiley, 2008.
Harvey, Richard A. Biochemistry (Lippincott's Illustrated
Reviews). 5th ed., 2010.
Boyer, Rodney F. Concepts in Biochemistry. 3rd ed., 2005.
2016A. Gluconeogenesis Trans
Madarcos Notes

th

[Devlin, 7 Ed]

Net synthesis or formation of glucose from noncarbohydrate substrate


Essential for survival of humans and other animals
as blood glucose levels have to be maintained to
support metabolism of tissues that use glucose as
their primary substrate
Enables the maintenance of blood glucose levels
long after all dietary glucose has been absorbed
and completely oxidized and glucose stored as
glycogen has been used up
SOURCES OF CARBON: amino acids, lactate,
pyruvate, propionate, and glycerol
Glucose can also be formed from fructose

th
[Harper, 29 Ed]

II.

Legend: Italicized quoted from the lecturer; bold


emphasis, or from references

- [1 of 12] -

Process of synthesizing glucose or glycogen form


non-carbohydrate precursors
MAJOR TISSUES: liver and kidney (kidney may
contribute up to 40% of total glucose synthesis in
fasting state and more in starvation)
Failure of gluconeogenesis is usually fatal
Important in maintaining the level of intermediates
of the citric acid cycle
Clears lactate produced by muscle and
erythrocytes and glycerol produced by adipose
tissue
IMPORTANT CYCLES BETWEEN TISSUES THAT
INVOLVE GLUCONEOGENESIS

[BIOCHEM]

OVERVIEW OF GLUCONEOGENESIS

Two cycles are critically important for maintaining


blood glucose levels
Depend on gluconeogenesis in liver followed by
delivery of glucose and its use in peripheral tissue
Provide a continuous supply of glucose to
tissue that require it as their primary energy source
To participate in the cycles, peripheral tissues must
release either alanine or lactate as the end product
of glucose metabolism

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


A.

CORI CYCLE (GLUCOSE LACTATE CYCLE)

2 ATP per glucose molecule in peripheral tissue


Upon oxidation of glucose in the muscle under
anaerobic glycolysis, lactate is produced.
The muscle does not contain the key enzymes of
gluconeogenesis so it would have to export lactate
through the blood to the liver and in the
hepatocytes lactate will be formed back to glucose.
Some glucose produced by the liver will be
returned to the muscle also to brain and RBC

III. PATHWAYS OF GLUCOSE SYNTHESIS

A.

Glucogenic
Glycine
Serine
Valine
Histidine
Arginine
Cysteine
Proline
Hydroxyproline
Alanine
Glutamate
Glutamine
Aspartate
Asparginine
Methionine

Cori cycle (RBC Liver RBC)

ALANINE CYCLE (GLUCOSE ALANINE CYCLE)

B.

GLUCONEOGENESIS FROM AMINO ACIDS

Most common source of glucose if amino acid is


the precursor
NADH generated by glycolysis is not used to
reduced pyruvate to lactate in this cycle; if it did,
pyruvate would not be available for conversion to
alanine by transamination with glutamate
Transfers energy from liver to peripheral tissue
Energetically more efficient because of the 6 to 8
molecules of ATP produced per molecule of
glucose
Through the action of alanine transaminase, the
carbon skeleton will be converted to pyruvate then
pyruvate will serve as precursor for glucose while
the amino group will be converted to urea then
exported to the blood and excreted through the
kidneys
Alanine transaminase enzyme is one of the
enzymes used to gauge the function of the liver,
the other enzyme is aspartate transaminase

Ketogenic
Leucine
Lysine

Both
Threonine
Isoleucine
Phenylalanine
Tyrosine
Tryptophan

Pyruvate through transamination can be converted


to glucose; the some amino acids through
transamination can be converted to pyruvate
All amino acids except leucine and lysine can
supply carbon for net synthesis of glucose by
gluconeogenesis
If catabolism of amino acids produces either
pyruvate or oxaloacetate, net glucose synthesis
can occur from that amino acid
Oxaloacetate
is
an
intermediate
in
gluconeogenesis and pyruvate is readily converted
to oxaloacetate by pyruvate carboxylase
Catabolism of amino acid feed carbon into the TCA
cycle at several points. Either through oxaloacetate
or through pyruvate
Reactions that lead to net synthesis of TCA cycle
intermediates are called anaplerotic reactions and
support gluconeogenesis because they provide for
net synthesis of oxaloacetate
Reactions catalyzed by pyruvate carboxylase and
glutamate dehydrogenase are good examples of
anaplerotic reactions
Leucine and lysine are ketogenic but not
glucogenic
Ketogenic: yields ketone body acetoacetate or
acetyl CoA
Glucogenic: yields either pyruvate or oxaloacetate
so can form glucose over again.
No pathway exists that can convert acetoacetate or
acetyl CoA to either pyruvate or oxaloacetate
because the conversion of pyruvate to acetyl
CoA is irreversible

B. GLUCONEOGENESIS FROM TRIACYLGLYCEROL

Hydrolysis of TAG yields three fatty acids and glycerol.


a.

Alanine cycle (Muscle Liver Muscle)


- [2 of 12] -

[BIOCHEM]

Glucose from Fatty Acids


Most fatty acids in humans have straight chains
with an even number of carbon atoms, their
catabolism yields acetyl CoA which cannot be
converted to oxaloacetate or any other

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat

b.

intermediates of gluconeogenesis. It is therefore


impossible to synthesize glucose from fatty
acids.
An exception to this rule applies to fatty acids
with methyl branches and fatty acids with an
odd number of carbon atoms, their catabolism
yields propionyl CoA which is a good precursor of
gluconeogenesis as it yields oxaloacetate by
anaplerotic pathway
Glucose from Glycerol
Phosphorylation of glycerol at carbon 3 by glycerol
kinase produces glyceraldehyde 3-phosphate
which is converted by glycerol phosphate
dehydrogenase enzyme at carbon 2 to DHAP. So
glycerol will enter gluconeogenic pathway as DHAP
+
Glycerol
requires
NAD
as
it
enters
gluconeogenesis
Reversible process, so DHAP can be converted
to glycerol and glycerol can be formed into TAG.
Glycerol comes also from the sugar fructose. High
fructose from fruit juices and sodas can force liver
to increase its ability to oxidize glucose and to
synthesize fatty acids, so increase TAG and
cholesterol

GALACTOSE

To be further metabolized, galactose must be


phosphorylated first by galactokinase to produce
galactose-1-phosphate

Galactose-1-phosphate, through the action of


galactose-1-uridyltransferase, can be converted to
UDP-galactose

UDP-galactose can be converted to glucose-1phosphate by UDP-galactose-4-epimerase followed


by UDP-glucose pyrophosphorylase

Phosphoglucomutase acts on glucose-1-phosphate


to convert it to glucose-6-phosphate

Glucose-6-phosphate can either enter glycolysis or


be converted in the liver by glucose-6-phosphatase
to glucose.
MANNOSE

D-mannose can be phosphorylated by hexokinase


and then converted to fructose-6-phosphate by
mannose phosphate isomerise

Fructose-6-phosphate may enter as a substrate of


glycolysis or gluconeogenesis.
IV. METABOLIC STEPS OF GLUCONEOGENESIS

Glucose from Glycerol


C.

GLUCONEOGENESIS FROM OTHER SUGARS

FRUCTOSE

Fructose has its own fructokinase

In liver, fructose is phosphorylated in carbon 1 by


fructokinase which yields fructose 1-phosphate

Fructose-1-phosphate has its own aldolase enzyme

Aldolase B cleaves fructose-1-phosphate between


carbon 3 and 4 to yield dihydroxyacetone
phosphate or DHAP (a 3 carbon intermediate which
is a glucose precursor) and D-glyceraldehyde (a 2
carbon intermediate)

Two molecules of DHAP from one molecule of


fructose can be converted to glucose by enzymes
of gluconeogenesis or into pyruvate or lactate by
last stage of glycolysis

- [3 of 12] -

[BIOCHEM]

Glycolysis and Glycogenolysis

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


#
1

Reaction
Pyruvate + CO2 + ATP Oxaloacetate + ADP + Pi

MALATE is transported from the mitochindria to the


cytosol
MALATE is reoxidized to OXALOACETATE in the
cytosol with the production of cystolic NADH

Oxaloacetate + GTP PEP + CO2 + GDP

PEP + H2O 2phosphoglycerate

2-phosphoglycerate 3-phosphoglycerate

3-phosphoglycerate + ATP 1,3-biphosphoglycerate + ADP

1,3-biphosphoglycerate + NADH + H Glyceraldehyde-3+


PO4 + NAD + Pi

Glyceraldehyde-3-PO4 Dihydroxyactone-PO4

Glyceraldehyde-3-PO4 + Dihydroxyactone-PO4 Fructose


1,6-biphosphate

L-malate + NAD Oxaloacetate + NADH + H


+

Fructose 1,6-biphosphate + H2O Fructose-6-PO4 + Pi

9
10

Fructose-6-PO4 Glucose-6-PO4

11

Glucose-6-PO4 + H2O Glucose + Pi

Step 1: Conversion of Pyruvate to Oxaloacetate


Conversion of Oxaloacetate Malate Oxaloacetate
Step 2: Conversion of Oxaloacetate to PEP

Enzyme: Pyruvate carboxylase

Mitochondrial enzyme
Requires biotin as coenzyme
First regulatory enzyme in the gluconeogenic
pathway
Requires acetyl-CoA as a positive effector
Note: Acetyl-CoA is produced by fatty acid
oxidation and its accumulation signals the
availability of fatty acids as fuel

Pyruvate is transported into the mitochondria or is


generated by alanine within the mitochondria by
transamination
o Transamination- -amino group is removed from
alanine (leaving pyruvate) and added to an -keto
carboxylic acid
Pyruvate is converted to oxaloacetate by pyruvate
carboxylase inside the mitochondria
Pyruvate carboxylase catalyzes the carboxylation of
pyruvate (3 carbons) to oxaloactate (4 carbons)
Mitochondrial membrane has no transporter for
oxaloacetate thus it is reduced to MALATE by MALATE
DEHYDROGENASE at the expense of NADH
Oxaloacetate + NADH + H L-malate + NAD
+

Enzyme: Phosphoenolpyruvate carboxykinase (PEPCK)

present in both cytosol and mitochondria matrix


Two ways of forming PEP:
1. Mitochondrial PEPCK may convert oxaloacetate to PEP
and be transported to the cytosol OR
2. Reduction of oxaloacetate to malate so as it can cross
the mitochondrial membrane and converting malate to
oxaloacetate again to be converted to PEP
2+

It is a Mg dependent reaction

PEPCK catalyzes phosphorylation of one molecule


of pyruvate to PEP using 2 molecules of GTP or
ATP (whereas in glycolysis only 1 ATP is produced
in the conversion of PEP to pyruvate). It also
removes the CO2 (decarboxylate) that is added to
the pyruvate carboxylase step to activate pyruvate
so that the oxaloacetate facilitates PEP formation.

In the liver and kidneys, GTP is produced from the


conversion of succinyl-CoA to succinate by succinyl
thiokinase via the TCA. This GTP can be used up
in gluconeogenesis thus establishing link in TCA
and gluconeogenesis.

- [4 of 12] -

[BIOCHEM]

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


Step 3: Conversion of Phosphoenolpyruvate to 2phosphoglycerate

Step 6: Conversion of 1,3-bisphosphoglycerate to


glyceraldehyde 3-phosphate

Enzyme: Enolase

Located in the cytosol and involved in glycolysis


and gluconeogenesis

Needs one mole of H2O per mole of phosphoenol


pyruvate

Enzyme: Glyceraldehyde-3-phosphate dehydrogenase

Located in the cytosol and involved in glycolysis


and gluconeogenesis
+

Tthis step oxidizes NADH to NAD to convert 1,3bisphosphoglycerate


to
glyceraldehyde-3phosphate

Step 4: Conversion
phosphoglycerate

3-

Step 7: Conversion of Glyceraldehyde-3-phosphate to


Dihydroxyacetone phosphate (DHAP)

Enzyme: Phosphoglycerate mutase

Located in the cytosol and involved in glycolysis


and gluconeogenesis

Enzyme: Triose phosphate isomerase

Located in the cytosol and involved in glycolysis


and gluconeogenesis

Step 5: Phosphorylation of 3-phosphoglycerate to 1,3bisphosphoglycerate

Step 8: Production of fructose 1,6 bisphosphate from


glyceraldehyde 3-phosphate

Enzyme: Phosphoglycerate Kinase

Located in the cytosol and involved in glycolysis


and gluconeogenesis

This step ultilizes ATP to phosphorylate 3phosphoglycerate to 1,3-bisphosphoglycerate

Enzyme: Aldolase A

Located in the cytosol and involved in glycolysis


and gluconeogenesis

of

2-phosphoglycerate

to

- [5 of 12] -

[BIOCHEM]

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


Step 9: Conversion of Fructose 1,6 bisphosphate to
Fructose 6- phosphate

Step 11: Dephosphorylation of Glucose 6-phophate to


glucose

Enzyme: Fructose-1,6-bisphosphatase

Enzyme releases Pi from fructose 1,6 phosphate to


form fructose 6-phophate

Enzyme: Glucose 6-phosphatase

Note: This is not a reversal of PFK-1 reaction; ATP is not


produced when phosphate group is removed from C1
because that is a low phosphate bond.

It is an allosteric enzyme that participates in the


regulation of gluconeogenesis
o Can be allosterically inhibited by fructose 2,6bisphosphate and AMP
o Can be allosterically stimulated by citrate

Present in the liver and skeletal muscle while


absent in heart and smooth muscle

Located in the cytosol

Hydrolysis of fructose-1,6-bisphosphate to fructose6-phosphate

Note: Glucose produced by gluconeogenesis in the liver and


kidney or ingested in the diet is delivered to brain and
muscle through blood stream

Absent in muscle and adipose tissue, thus they


cant
export
glucose
into
the
blood
streamHydrolysis of phosphate ester:
Glucose 6-phosphate + H2O glucose + Pi

Step 10: Conversion of Fructose 6-phosphate to glucose


6- phosphate

G6P is hydrolyzed as it passes into the ER. ER


vesicle filled with glucose diffuse to plasma
membrane, fuse with it and open, releasing glucose
into the blood stream

Action of Glucose-6-Phosphatase in Hepatocytes


(Endoplasmic Reticulum)

Enzyme: Phosphoglucose isomerase

Located in the cytosol and involved in glycolysis


and gluconeogenesis

V.
1.

- [6 of 12] -

[BIOCHEM]

Enzyme is found in on the lumenal side of the


endoplasmic reticulum of hepatocytes and renal
cells. Muscle and brain tissue do not contain this
enzyme and so cant carry out gluconeogenesis

GLUCONEOGENIC ENZYMES

Pyruvate Carboxylase

Pyruvate Oxaloacetate Step1 of 2

Mitochondrial enzyme, anaplerotic

First regulatory enzyme in the gluconeogenic


pathway
o Coenzyme: Biotin
Energy requiring reaction
Biotin binds CO2 from bicarbonate as carboxybiotin
prior to addition of the CO2 to pyruvate
Requiring acetyl--CoA as a positive effector
produced by fatty acids oxidation

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


Phosphoenolpyruvate Carboxykinase (PEPCK)

OxaloacetatePhosphoenolpyruvate Step2 of 2

Cytosolic/mitochondrial enzyme

Requires 2 mol of GTP (from a reaction in the citric


acid cycle) to convert to 2 mol of oxaloacetate to 2
mol of PEP

Mitochondrial PEPCK - converts oxaloacetate to PEP and


be transported to the cytosol

2.

4.

Cytosolic PEPCK

Inducible enzyme (the quantity of the enzyme in


the cell increases because of increased
transcription of its gene and increased translation
of its mRNA)

Cytosolic carboxylase converts oxaloacetate to


aspartate first, which exits mitochondrion by way of
glutamate-aspartate antiport. Aspartate contributes
its amino group to -ketoglutarate in the cytosol to
produce oxaloacetate, which is used by cytosolic
PEP carboxykinase to produce PEP

Acts on the bisphosphate to release inorganic


phosphate
Present in liver, kidney, and skeletal muscle
Fructose 2,6-bisphosphate and AMP are the
allosteric inhibitor (when the allosteric effectors
are low, PFK-1 is less active, F 1,6-BPase is
more active)
Induced during fasting

Glucose 6-Phosphatase
Glucose 6 phosphate Glucose
Induced during fasting
In luminal side of the ER
Present in liver and kidney
Absent in muscle, adipose and brain tissue,
therefore cannot export glucose into the
bloodstream
2+
Mg enzyme found on the luminal side of the
endoplasmic reticulum of hepatocytes and renal
cells
Enzyme

Positive
effectors

Negative
effectors

PC/PEPCK

Acetyl CoA

F 1,6-BP

F 1,6-BPase

F 2,6-BPase,
AMP

G6Pase

F6P
VI. REGULATION
A.

EXPENDITURE OF ATP

Gluconeogenesis requires expenditure of ATP

Synthesis of one glucose from lactate = 6 ATPs

Synthesis of one glucose from alanine = 10 ATPs

Mechanism of Cytosolic PEPCK (Route 1) (ketoacid = -ketoglutarate)


cAMP(Cyclic adenosine monophosphate) is the major
inducer wherein it activates protein kinase A

Induced
by
glucagon,
epinephrine
and
glucocorticoids

Glucagon increases cAMP during fasting

Epinephrine acts during exercise/stress

Repressed by insulin

B.

3.

Fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphate Fructose 6


phosphate

Removes phosphoryl group in C1

REGULATION OF GLUCONEOGENESIS

Active: gluconeogenesis, inhibited: glycolysis


Inhibition of glycolysis at its chief regulatory sites/
repression of enzymes that bypass these sites=
increase effectiveness of gluconeogenic enzymes

Fatty acid oxidation supplies requirements needed by


gluconeogenesis:
1. ATP
2. Increased concentration of acetyl CoA
3. Increased concentration of NADH
These products activates pyruvate dehydrogenase
kinase which

Phosphorylates
and
inactivates
pyruvate
dehydrogenase complex

Prevents pyruvate from being converted to acetyl


CoA; conservation of pyruvate for glucose synthesis

- [7 of 12] -

[BIOCHEM]

ATPs for glucose synthesis are mostly provided by fatty


acid oxidation
Synthesis of glucose in liver favor increased availability
of fatty acids in the blood oxidized by liver
mitochondria to ketone bodies with ATP production.

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


ACETYL CoA

Positive allosteric effector of pyruvate


carboxylase

Directs C of pyruvate to OAA for glucose synthesis

Increase in OAA due to increased pyruvate


carboxylase activity + increased acetyl CoA from
fatty acid oxidation = greater synthesis of citrate
CITRATE

Negative allosteric effector of 6-phosphofructo-1kinase and pyruvate kinase

Inhibition of 6-phosphofructo-1-kinase decreases


concentration of F 1,6-bisphosphate, an activator of
pyruvate kinase

Inhibition of pyruvate kinase


o Decreases flux of PEP to pyruvate
o Increases
effectiveness
of
pyruvate
carboxylase and PEP carboxykinase coupling
for pyruvate to PEP conversion
INCREASE IN ATP LEVELS, DECREASE IN AMP
LEVELS

Favors gluconeogenesis

Inhibition of 6-phosphofructo-1-kinase and pyruvate


kinase

Activation of F1,6-BPase

C.

HORMONAL REGULATION

Regulation of supply of fatty acids to liver


Regulation of enzyme activities of both glycolysis
and gluconeogenesis

CATECHOLAMINES

Increase plasma fatty acids by promoting lipolysis


in adipose tissue

Action is opposed by insulin (inhibits lipolysis)


GLUCAGON

Activates adenylate cyclase to produce cAMP

Activation of protein kinase A follows


o pkA phosphorylates pyruvate kinase
INACTIVE

Gluconeogenesis is stimulated:
o By blocking conversion of PEP to pyruvate
o Decreased concentration of fructose 2,6bisphosphate (allosteric effector of 6phosphofructo-1-kinase; allosteric inhibitor of
Fructose 1,6-BPase)

ACTIVATION
OF
GLYCOLYSIS
(LIVER:
FROM
GLUCONEOGENESIS TO GLYCOLYSIS)

Shortage of oxygen for respiration

Shortage of fatty acids for oxidation

Inhibition
or
uncoupling
of
oxidative
phosphorylation

Glucagon effect on Pyruvate Kinase


6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE 2,6BISPHOSPHATASE

Regulatory mechanisms of glycolysis and gluconeogenesis.


(+) activators. (-) inhibitors

- [8 of 12] -

[BIOCHEM]

Bifunctional enzyme
Phosphorylation of this enzyme is stimulated by
cAMP
When phosphorylated
o Kinase activity: INACTIVE
o Phosphatase activity: ACTIVE
Fructose-2,-6-Bisphosphate
o Potent stimulator of phosphofructokinase
o Powerful
inhibitor
of
fructose-1,6bisphosphatase

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


o

Inhibition occurs in either the presence or


absence of AMP
o Effects of AMP and fructose-2,6-bisphosphate
are synergistic
Lowers fructose 2,6-bisphosphate concentration
o Leads
to
decreased
activity
of
6phosphofructo-1-kinase
o F1,6-bisphosphatase=more active
Overall effect:
o Increased conversion of F-1,6-BP to F6P
o Increase in rate of gluconeogenesis
o Increase in fructose-6-phosphate: inhibition of
glucokinase via its inhibitory protein

INSULIN

Signals activation of cAMP phosphodiesterase

Inhibits protein kinase A

Activates phosphoprotein phosphatase


GLUCAGON/INSULIN RATIO IN BLOOD (see appendix)

Increases when gluconeogenesis is needed

Decreases when glucose from GIT is plenty


o High
ratioincreased
capacity
of
gluconeogenesis, decreased glycolysis in liver
o Low
Ratio-decreased
capacity
of
gluconeogenesis, increased glycolysis in liver
GLUCAGON REGULATION

Glucagon binds to plasma membrane receptor:


increase in cAMP; protein kinase A: ACTIVATED

PKA phosphorylates cAMP-response element


binding protein (CREB)

CREB-P (transcription factor) binds to cAMPresponse element (CRE)


o CRE-cis acting element w/in regulatory region
of genes responsive to cAMP

Promotes transcription of genes for PEPCK

Glucagon also repress transcription results to


decreased amounts of the ff:
o Glucokinase
o 6-phosphofructo-1-kinase/fructose-1,6-BPase
o Pyruvate kinase

Decreased glucagon/insulin ratio in blood


decreases transcription of gluconeogenic enzymes,
increases transcription of glycolytic enzymes

Glucagon promotes transcription of the gene that


encodes PEPCK
ALCOHOLIC OXIDATION

Inhibits gluconeogenesis

Oxidation of alcohol (ethanol) by liver produces


large load of reducing equivalents (NADH) that
have to be transported into the mitochondria by
malate-aspartate shuttle

Excess NADH in the cytosol-forces equilibrium of


reactions catalyzed by lactate dehydrogenase
(lactate formation) and malate dehydrogenase
(malate formation)

Gluconeogenesis is inhibited by limiting availability


of pyruvate and OAA for pyruvate carboxylase and
PEPCK reactions, respectively.

Alcohol inhibition in gluconeogenesis


D.

FATES OF LACTATE FORMED DURING MUSCLE


CONTRACTION

LACTATE

Produced by skeletal muscle cells and RBCs

Source of energy by other organs

Released in the blood from anaerobic glycolysis in


skeletal muscles and RBCs and other cells that
lack mitochondria or have low O2 concentration
taken by the liver and the kidney converted first
to pyruvate via lactate dehydrogenase enters
the mitochondria converted to oxaloacetate
glucose
- [9 of 12] -

[BIOCHEM]

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


TWO FATES OF LACTATE
1. Plasma membrane of some cells (e.g. Cardiac
muscle cells) contains carriers with high
permeability to lactate and pyruvate.

Once inside well-oxygenated cells, lactate


can be reverted back to pyruvate and
metabolized through TCA and Ox Phos to
produce ATP

Use of lactate makes more circulating


glucose available to the active muscle
cells
2. Excess Lactate enters the liver

Lactate in the liver is converted to


pyruvate first then into glucose through
gluconeogenesis

Constitutes the Cori cycle


E.

PEPCK REGULATION IN PROMOTER REGION


CREB turns on the synthesis of glucose 6phosphatase and PEP carboxykinase
FOXO1 (forkhead box other) stimulates
synthesis
of
gluconeogenesis
enzymes,
suppresses synthesis of enzymes of glycolysis,
pentose phosphate pathway, and triaclyglycerol
synthesis
o Unphosphorylated form FOXO1 acts as
nuclear transcription factor
o Response to insulin:
FOXO1 leaves the nucleus into the cytosol,
wherein it is phosphorylated by PKB then
tagged by ubiquitin and degraded by
proteasome
o Glucagon
Glucagon prevents phosphorylation by PKB,
thus, FOXO1 remains active in the nucleus
Gene encoding for PEP carboxykinase
o With multiple transcription factors that act on the
same promoter activated by multiple protein
kinases and phosphatases with variety of
accessory factors for modulation
o 15 or more response elements in promoter
region
o Mutations in transcription factors affect
regulation

Mechanism of FOXO1 in gene transcription and translation


VII. SUMMARY OF GLUCONEOGENESIS
1.

2.
3.
4.

Enzyme
Pyruvate
carboxylase and
PEP
carboxykinase
(PEPCK)
Fructose-1,6Bisphosphatase
Glucose-6phosphatase
5.

6.
7.
- [10 of 12] -

[BIOCHEM]

Gluconeogenesis is the generation of new glucose.


In addition to pyruvate, lactate, and other hexoses,
other noncarbohydrate precursors can be tapped
as substrates for gluconeogenesis including most
of the amino acids, as well as glycerol and all the
TCA cycle intermediates
Lysine and leucine are the only amino acids that
are not substrates for gluconeogenesis.
The major sites of glucogeneogenesis are the liver
and kidneys (90% liver, 10% kidney)
The conversion of pyruvate to glucose by
gluconeogenesis requires enzymes that bypass the
three exergonic steps of glycolysis:
Enzyme Bypassed

Step # in
Glycolysis

Pyruvate kinase

10

Phosphofructokinase
(PFK)

Hexokinase/Glucokinase

Conversion of glucose-6-phosphate glucose


occurs only in hepatocytes due to the presence of
glucose-6-phosphatase. Skeletal muscles do not
convert glucose-6-phosphate glucose due to the
absence of the enzyme
Control of gluconeogenesis can be done through
allosteric regulation or through hormone signalling
The ratio of glucagon to insulin through the
increase/decrease in cAMP production can affect

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat

8.

9.

the activation/inactivation of the bifunctional


enzyme complex of 6-phosphofructokinase-2 (PFK2) and fructose-2,6-bisphosphatase
Transcription factors, like FOXO1 and CREB, which
act on the promoter region of enzymes, can affect
the rate of gluconeogenesis by increasing the gene
expression of specific regulatory enzymes.
Mutations in transcription factors affect regulation
and can lead to diseases.
VIII. APPENDIX

Effect of elevated glucagon on the intracellular concentration of fructose-2,6-bisphosphate in the liver


Gluconeogenesis: Regulation
PEPCK promoter region
Gene Expression
Process
Replication
Transcription
Translation

Parent Compound

Product

DNA
DNA
RNA

DNA
DNA
Protein

Conditions

Activation

Inhibition

Pathway active

High AMP
concentration

Phosphofructokinase

Fructose 1,6 - BPase

Glycolysis

ATP and Citrate

Phophofructokinase

Gluconeogenesis

ATP and Alanine

Pyruvate Kinase

Gluconeogenesis

ADP

Pyruvate
Carboxylase
PEP Carboxykinase

Glycolysis

Acetyl CoA

Fructose 1,6 BPase

Pyruvate
Carboxylase

Gluconeogenesis

- [11 of 12] -

[BIOCHEM]

Intracellular
Location
Nucleus
Nucleus
Cytoplasm
Additional
Information

Citrate is an indicator
of the status of TCA
which means energy
charge
is
high,
biosynthetic
intermediates
are
abundant
(for
gluconeogenesis)
Energy charge is
high,
Biosynthetic
intermediates
are
abundant.

Like
Citrate
(indicates status of
TCA)

[BIOCHEM] 2.7 Gluconeogenesis Dr. Magat


1.

2.

IX. STUDY GUIDE


Which of the following is NOT a characteristic of gluconeogenesis?
A. It requires energy in the form of ATP and GTP
B. It is important in maintaining blood glucose during the normal overnight fast
C. It uses carbon skeletons provided by degradation of amino acids
D. It consists of all the reactions of glycolysis functioning in the reverse direction
E. It involves the enzyme fructose-1,6-bisphosphatase
Which of the following supports gluconeogenesis?
A.

-ketoglutarate + aspartate glutamate + oxaloacetate

B.

pyruvate + ATP + HCO3 oxaloacetate + ADP + Pi + H

C.
D.

acetyl-CoA + oxaloacetate + H2O citrate + CoA


lysine degradation

3.

The uncontrolled production of NADH from NAD during ethanol metabolism blocks gluconeogenesis from all of the
following EXCEPT:
A. galactose
B. glycerol
C. -ketoglutarate
D. oxaloacetate

4.

Which of the following statements concerning gluconeogenesis is correct?


A. It occurs in the muscle
B. It is stimulated by fructose-2,6-bisphosphate
C. It is inhibited by elevated levels of acetyl-CoA
D. It is important in maintaining blood glucose during the normal overnight fast
E. It uses carbon skeletons provided by degradation of fatty acids

5.

Elevated levels of circulating glucagon are associated with which one of the following:
A. Increased activity of phosphofructokinase-2
B. Decreased level of fructose-2,6-bisphosphatase
C. Decreased fructose-1,6-bisphosphatase activity
D. Fasting
E. Ingestion of a carbohydrate-rich meal

6.

Which of the following is NOT a glucogenic amino acid


A. Aspartate
B. Glutamine
C. Lysine
D. Glycine

Answers: D, B, A, D, D, C

- [12 of 12] -

[BIOCHEM]