Post on 25-May-2015
Gluconeogenesis - Introduction
Gluconeogenesis: The synthesis of glucose from noncarbohydrate
precursors (e.g., lactate , pyruvate, glycerol, citric acid cycle
intermediates, amino acids).
•Glucose is the major fuel source for the brain, nervous system,
testes, erythrocytes, and kidney medulla.
•Daily requirement: 160 grams.
•Approx. 20 grams of glucose is present in body fluids.
•Approx. 190 grams is available as stored glycogen.
•Thus sufficient reserves for 1 day’s requirement.
•During starvation or intense exercise, glucose must be replaced by
gluconeogenesis.
•Major site of gluconeogenesis: Liver
•Secondary site: Kidney cortex.
•Thus gluconeogensis in the liver and kidney helps to maintain the
glucose level in the blood so that brain and muscle can extract
sufficient glucose to meet their metabolic demands.
Entry of Noncarbohydrate Precursors
Pyruvate Glucose
Seven out of ten reactions
of gluconeogenesis are
exact reversals of
glycolysis.
Three steps in glycolysis
are irreversible and thus
cannot be used in gluco-
neogenesis.
Therefore there are 3 steps
for which bypass reactions
are needed.
HK
PFK
PK
Note places of entry
of noncarbohydrate
precursors.
PEP
Pyruvate
Glucose
Glucose 6-phosphate
Fructose 6-phosphate
Fructose 1,6-bisphosphate
First Bypass Reaction: Convervsion of Pyruvate to
Phosphoenolpyruvate
Requires participation of both mitochondrial and cytosolic enzymes.
Step 1: Pyruvate is transported from the cytosol into mitochondria via
the mitochondrial pyruvate transporter OR pyruvate may be generated
within mitochondria via deamination of alanine.
Step 2: Pyruvate is converted to OAA by the biotin-requiring enzyme
pyruvate carboxylase as follows:
Pyruvate + HCO3- + ATP oxaloacetate + ADP + Pi + H+
Pyruvate carboxylase is a regulatory enzyme. Acetyl CoA is a
positive effector.
Mitochondria are the
source of reducing
equivalents that will
be needed later.
Mitochondrial
Malate dehydrog.
Pyruvate
transporter
Malate/α-KG
transporter
Produced in muscle
Or RBCs
Step 3: Oxaloacetate is reduced to malate by mitochondrial malatedehydrogenase at the expense of mitochondrial NADH.
Oxaloacetate + NADH + H+ L-malate + NAD+
Step 4: Malate exits the mitochondrion via the malate/α-ketoglutarate
carrier.
Step 5: In the cytosol, malate is reoxidized to oxaloacetate via
cytosolic malate dehydrogenase with the production of cytosolic
NADH.
L-malate + NAD+ oxaloacetate + NADH + H+
Mitochondria are the
source of reducing
equivalents that will
be needed later.
Mitochondrial
Malate dehydrog.
Pyruvate
transporter
Malate/α-KG
transporter
Produced in muscle
Or RBCs
Step 6: Oxaloacetate is then converted to phosphoenolpyruvate (PEP)
by phosphoenolpyruvate carboxykinase in the reaction:
Oxaloacetate + GTP phosphoenolpyruvate + CO2 + GDP
The overall equation for this set of bypass reactions is:
Pyruvate + ATP + GTP + HCO3-
phosphoenolpyruvate + ADP + GDP + Pi + H+ + CO2
Thus the synthesis of one molecule of PEP requires an investment
of 1 ATP and 1 GTP.
Note: when either pyruvate or the ATP/ADP ratio is high, the reaction
is pushed toward the right (i.e., in the direction of biosynthesis).
Mitochondria are the
source of reducing
equivalents that will
be needed later.
Mitochondrial
Malate dehydrog.
Pyruvate
transporter
Malate/α-KG
transporter
Produced in muscle
Or RBCs
When lactate is the gluconeogenic precursor (e.g. after vigorous
exercise) an abbreviated pyruvate to PEP bypass is utilized.
Important Point: Conversion of lactate to pyruvate (via LDH) in the
cytosol yields NADH which is essential for gluconeogenesis to
proceed (i.e., NADH is needed at the glyceraldehyde 3-phosphatedehydrogenase step). Thus, the export of malate from mitochondria
is no longer necessary as a source of NADH. In the abbreviated
pathway:
Step 1: Pyruvate is transported into mitochondria on the pyruvate
transporter.
Step 2: Within mitochondria pyruvate is converted to OAA (via
pyruvate carboxylase).
Step 3: Intramitochondrial oxaloacetate is converted to PEP (via a
mitochondrial form of PEP carboxykinase).
Step 4: PEP is transported out of mitochondria and continues up the
gluconeogenic pathway.
Mitochondria are the
source of reducing
equivalents that will
be needed later.
Mitochondrial
Malate dehydrog.
Pyruvate
transporter
Malate/α-KG
transporter
Produced in muscle
or RBCs
Mitochondrial
Malate dehydrog.
Pyruvate
transporter
Malate/α-KG
transporter
Produced in muscle
or RBCs
How do mito sense
which pathway to
follow?
High cyt. lactate
High cyt. NADH
High cyt. malate
High mit. malate
High mit. OAA
Shunts OAA into
PEP production
in mito.
PEP
Pyruvate
Glucose
Glucose 6-phosphate
Fructose 6-phosphate
Fructose 1,6-bisphosphate
Second Bypass Reaction: Conversion of Fructose 1,6-
bisphosphate to Fructose 6-phosphate
The second glycolytic reaction (i.e., the phosphorylation of fructose
6-phosphate by PFK1) is irreversible.
Hence, for gluconeogenesis fructose 6-phosphate must be generated
from fructose 1,6-bisphosphate by a different enzyme:
fructose 1,6-bisphosphatase.
This reaction is also irreversible.
Fructose 1,6-bisphosphate + H2O fructose 6-phosphate + Pi
ΔG˚’ = -3.9 kcal/mol
PEP
Pyruvate
Glucose
Glucose 6-phosphate
Fructose 6-phosphate
Fructose 1,6-bisphosphate
Third Bypass Reaction: Glucose 6-phosphate to Glucose
Because the hexokinase reaction is irreversible, the final reaction of
gluconeogenesis is catalyzed by a different enzyme, namely
glucose 6-phosphatase.
Glucose 6-phosphate + H2O glucose + Pi
ΔG˚’ = -3.3 kcal/mol
Glucose 6-phosphatase is present in the liver, but absent in brain and
muscle. Thus, glucose produced by gluconeogenesis in the liver, is
delivered by the bloodstream to brain and muscle.*****
The overall equation for gluconeogenesis is:
2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 4 H2O
glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+
For each molecule of glucose produced, 6 high energy phosphate
groups are required as are 2 molecules of NADH.
Thus “Gluconeogenesis Costs”.
Gluconeogenesis from Various Metabolites
Citric Acid Cycle Intermediates: form oxaloacetate during one turn
of the cycle. Can get net synthesis of glucose from citric acid cycle
intermediates.
3 carbons of the resulting OAA are converted into glucose, 1 carbon
is released as CO2 by PEP carboxykinase.
Amino Acids: all can be metabolized to either pyruvate or certain
intermediates of the citric acid cycle. Hence they are glucogenic
(i.e., they can undergo net conversion to glucose). Exceptions are
leucine and lysine.
Alanine and glutamine are of special
importance as they are used to
transport amino groups from a variety
of tissues to liver deaminated to
pyruvate and α-KG gluconeogenesis.
Fatty Acids: even numbered carbon FA are not converted into
glucose since during catabolism they yield only acetyl CoA
which can’t be used as a glucose precursor.
Since: for every 2 carbons the enter the cycle as acetyl CoA, 2
carbons are lost as CO2, thus there is no net production of OAA
to support glucose biosynthesis.
FA oxidation does contribute in that it provides ATP and NADH
needed to fuel gluconeogenesis.
ADD TO HANDOUT:
In contrast odd numbered carbon FAs propionyl CoA
succinyl CoA which enters the cycle past the decarboxylation
steps.
Thus one can synthesize glucose from odd chain fatty acids.
Glycerol (which can be generated by hydrolysis of triacylglycerols
(fat) to yield free FAs + glycerol) is an excellent substrate for gluco-
neogenesis.
Gluconeogenesis Glycolysis
Substrate Cycles
A pair of reactions such as the phosphorylation of fructose 6-phosphate
to fructose 1,6-phosphate and its hydrolysis back to the starting material
is called a substrate cycle.
ATP + fructose 6-phosphate ADP + fructose 1,6-bisphosphate + H+
Fructose 1,6-bisphosphate + H2O fructose 6-phosphate + Pi
Typically, both reactions are not simultaneously fully active in the same
cell because of reciprocal regulation.
PFK1
Fructose 1,6-bisphosphatase
Substrate cycles can serve two functions:
1) Amplification of metabolic signals.
2) To generate heat which is released
during the hydrolysis of ATP.
ATP + H2O ADP + Pi + H+ + Heat
Assume an allosteric effector:
increases A B by 20% and
decreases B A by 20%;
Result is to increase the net
flux of B by 380%.
Reciprocal regulation is exquisitely
sensitive!!!
Gluconeogenesis and Glycolysis are Reciprocally Regulated
First Coordinated Control Point: Pyruvate PEP
(3) Pyruvate Carboxylase: stimulated by acetyl CoA. Inhibited by ADP.Thus when excess acetyl CoA builds up glucose formation is stimulated.
When the energy charge in the cell is low, biosynthesis is turned off.
(1) Pyruvate Kinase: inhibited by ATP and alanine. Activated by F-1,6-BP.Thus high energy charge or abundance of biosynthetic intermediates turn off glycolysis.
Glycolytic pathway intermediate turns it on.
(2) PEP Carboxykinase: ADP turns it off.Thus when energy charge of the cell is low, the biosynthetic pathway is turned off.
(1)
(2)
(3)
Finally, recall that PDH is inhibited by acetyl CoA. Thus excess acetyl CoA
slows its formation from pyruvate and stimulates gluconeogenesis by
activating pyruvate carboxylase.
Thus F-1,6-BPase is inhibited by F-2,6-BP and AMP.
These modulators have the opposite effect on PFK1.
Further, recall that F-2,6-BP is a signal molecule that is present at low
concentration during starvation and high concentration in the fed state
due to the antagonistic effects of glucagon and insulin on its production.
Second Coordinated Control Point:
Fructose 1,6-Bisphosphate Fructose 6-phosphate
•The activities of PFK2 and FBPase2 reside on the same polypeptide
chain.
•Both activities are reciprocally regulated by phosphorylation of a
single serine residue.
Thus low blood glucose, blood glucagon, cAMP-dependent
phosphorylation of this bifunctional enzyme, PFK2 and FBPase 2,
which then F 2,6-BP, and then PFK1 and Fructose 1,6-bis-
phosphatase.
Fructose 6-phosphate Fructose 2,6-bisphosphatePFK2
Fructose bisphosphatase 2
BOTTOM LINE: WHEN BLOOD GLUCOSE IS LOW:
GLYCOLYSIS AND GLUCONEOGENESIS
SO THAT YOU MAKE MORE GLUCOSE IN THE LIVER!!!!
Hormonal Regulation of Gluconeogenesis
Hormonal regulation occurs via 3 basic mechanisms:
1) Regulation of the supply of fatty acids and glycerol to the liver.
• Glucagon increases plasma fatty acids and glycerol by promoting
lipolysis in adipose tissue. This effect is antagonized by insulin.
• The result is increased fatty acid oxidation by the liver which
promotes gluconeogenesis via the generation of NADH, ATP,
acetyl CoA, and increased gluconeogenic substrate (glycerol).
2) Regulation of the state of phosphorylation of hepatic enzymes.
• Glucagon activates adenylate cyclase to produce cAMP, which
activates protein kinase A, which then phosphorylates and
INACTIVATES pyruvate kinase thereby decreasing glycolysis.
•Glucagon stimulates gluconeogenesis by decreasing the concentration
of F-2,6-BP in the liver.
Mechanism:
Glucagon adenylate cylase to produce cAMP, which then
a cAMP-dependent protein kinase to phosphorylate PFK2/fructose
bisphosphatase 2. This phosphorylation the kinase and
the phosphatase activity. Both of which lead to a in the F-2,6-BP
level.
Reduced F-2,6-BP leads to:
i) a decrease in PFK1 activity (and thus a decrease in glycolysis)
AND
ii) an increase in fructose 1,6-bisphosphatase activity (and thus
an increase in gluconeogenesis).
•Insulin causes the opposite effects.
3) Glucagon and insulin mediate long-term effects by inducing and
repressing the synthesis of key enzymes.
Glucagon induces the synthesis of:
PEP-carboxykinase
Gluconeogenic fructose 1,6-bisphosphatase
enzymes glucose 6-phosphatase
certain aminotransferases
Glucagon represses the synthesis of:
glucokinase
Glycolytic PFK1
enzymes pyruvate kinase
Insulin generally opposes these actions.
Thus a high glucagon/insulin ratio in the blood:
i) increases the enzymatic capacity for gluconeognesis
ii) decreases the enzymatic capacity for glycolysis
The Cori Cycle
Lactate and alanine, produced by skeletal muscle and RBCs are the
major fuels for gluconeogenesis.
Pyruvate lactate
alanine
LDH
The cycle in which part of the metabolic burden is shifted from the
muscle to the liver is known as the Cori Cycle.
Low NADH/NAD+
Ratio
& RBC
High NADH/NAD+
Ratio
AlanineAlanine