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Gluconeogenesis - Introduction
Gluconeogenesis: The synthesis of glucose from noncarbohydrateprecursors (e.g., lactate , pyruvate, glycerol, citric acid cycleintermediates, 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 days requirement.
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During starvation or intense exercise, glucose must be replaced bygluconeogenesis.
Major site of gluconeogenesis: Liver
Secondary site: Kidney cortex.
Thus gluconeogensis in the liver and kidney helps to maintain theglucose level in the blood so that brain and muscle can extract
sufficient glucose to meet their metabolic demands.
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Entry of Noncarbohydrate Precursors
Pyruvate Glucose
Seven out of ten reactionsof gluconeogenesis areexact reversals ofglycolysis.
Three steps in glycolysisare irreversible and thuscannot be used in gluco-neogenesis.
Therefore there are 3 stepsfor which bypass reactionsare needed.
HK
PFK
PK
Note places of entry
of noncarbohydrateprecursors.
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PEP
Pyruvate
Glucose
Glucose 6-phosphateFructose 6-phosphate
Fructose 1,6-bisphosphate
4
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First Bypass Reaction: Convervsion of Pyruvate toPhosphoenolpyruvate
Requires participation of both mitochondrial and cytosolic enzymes.
Step 1: Pyruvate is transported from the cytosol into mitochondria viathe mitochondrial pyruvate transporter OR pyruvate may be generatedwithin mitochondria via deamination of alanine.
Step 2: Pyruvate is converted to OAA by the biotin-requiring enzymepyruvate carboxylaseas follows:
Pyruvate + HCO3
- + ATP oxaloacetate + ADP + Pi + H+
Pyruvate carboxylaseis a regulatory enzyme. Acetyl CoA is apositive effector.
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Mitochondria are thesource of reducingequivalents that willbe needed later.
MitochondrialMalate dehydrog.
Pyruvatetransporter
Malate/-KGtransporter
Produced in muscle
Or RBCs
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Step 3: Oxaloacetate is reduced to malate by mitochondrial malatedehydrogenaseat the expense of mitochondrial NADH.
Oxaloacetate + NADH + H+ L-malate + NAD+
Step 4: Malate exits the mitochondrion via the malate/-ketoglutaratecarrier.
Step 5: In the cytosol, malate is reoxidized to oxaloacetate viacytosolic malate dehydrogenasewith the production of cytosolicNADH.
L-malate + NAD+ oxaloacetate + NADH + H+
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Mitochondria are thesource of reducingequivalents that willbe needed later.
MitochondrialMalate dehydrog.
Pyruvatetransporter
Malate/-KGtransporter
Produced in muscleOr RBCs
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Step 6: Oxaloacetate is then converted to phosphoenolpyruvate (PEP)by phosphoenolpyruvate carboxykinasein 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 reactionis pushed toward the right (i.e., in the direction of biosynthesis).
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Mitochondria are thesource of reducingequivalents that willbe needed later.
MitochondrialMalate dehydrog.
Pyruvatetransporter
Malate/-KGtransporter
Produced in muscleOr RBCs
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When lactate is the gluconeogenic precursor (e.g. after vigorousexercise) an abbreviated pyruvate to PEP bypass is utilized.
Important Point:Conversion of lactate to pyruvate (via LDH) in thecytosol yields NADH which is essential for gluconeogenesis toproceed (i.e., NADH is needed at the glyceraldehyde 3-phosphatedehydrogenasestep). Thus, the export of malate from mitochondriais no longer necessary as a source of NADH. In the abbreviatedpathway:
Step 1: Pyruvate is transported into mitochondria on the pyruvatetransporter.
Step 2: Within mitochondria pyruvate is converted to OAA (viapyruvate carboxylase).
Step 3: Intramitochondrial oxaloacetate is converted to PEP (via amitochondrial form of PEP carboxykinase).
Step 4: PEP is transported out of mitochondria and continues up thegluconeogenic pathway.
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Mitochondria are thesource of reducingequivalents that willbe needed later.
MitochondrialMalate dehydrog.
Pyruvatetransporter
Malate/-KGtransporter
Produced in muscle
or RBCs
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MitochondrialMalate dehydrog.
Pyruvatetransporter
Malate/-KGtransporter
Produced in muscleor RBCs
How do mito sensewhich pathway to
follow?
High cyt. lactate
High cyt. NADH
High cyt. malate
High mit. malate
High mit. OAA
Shunts OAA intoPEP productionin mito.
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PEP
Pyruvate
Glucose
Glucose 6-phosphateFructose 6-phosphate
Fructose 1,6-bisphosphate
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Second Bypass Reaction: Conversion of Fructose 1,6-bisphosphate to Fructose 6-phosphate
The second glycolytic reaction (i.e., the phosphorylation of fructose6-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
G = -3.9 kcal/mol
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PEP
Pyruvate
Glucose
Glucose 6-phosphateFructose 6-phosphate
Fructose 1,6-bisphosphate
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Third Bypass Reaction: Glucose 6-phosphate to Glucose
Because the hexokinase reaction is irreversible, the final reaction ofgluconeogenesis is catalyzed by a different enzyme, namelyglucose 6-phosphatase.
Glucose 6-phosphate + H2O glucose + Pi
G = -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.*****
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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.
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Gluconeogenesis from Various Metabolites
Citric Acid Cycle Intermediates: form oxaloacetate during one turnof the cycle. Can get net synthesis of glucose from citric acid cycle
intermediates.
3 carbons of the resulting OAA are converted into glucose, 1 carbonis released as CO2 by PEP carboxykinase.
Amino Acids: all can be metabolized to either pyruvate or certainintermediates of the citric acid cycle. Hence they are glucogenic(i.e., they can undergo net conversion to glucose). Exceptions areleucine and lysine.
Alanine and glutamine are of specialimportance as they are used to
transport amino groups from a varietyof tissues to liver deaminated topyruvate and -KG gluconeogenesis.
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Fatty Acids: even numbered carbon FA are not converted intoglucose since during catabolism they yield only acetyl CoAwhich cant be used as a glucose precursor.
Since: for every 2 carbons the enter the cycle as acetyl CoA, 2carbons are lost as CO2, thus there is no net production of OAAto support glucose biosynthesis.
FA oxidation does contribute in that it provides ATP and NADHneeded to fuel gluconeogenesis.
ADD TO HANDOUT:
In contrast odd numbered carbon FAs propionyl CoA
succinyl CoA which enters the cycle past the decarboxylationsteps.
Thus one can synthesize glucose from odd chain fatty acids.
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Glycerol (which can be generated by hydrolysis of triacylglycerols(fat) to yield free FAs + glycerol) is an excellent substrate for gluco-neogenesis.
Gluconeogenesis Glycolysis
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Substrate Cycles
A pair of reactions such as the phosphorylation of fructose 6-phosphateto fructose 1,6-phosphate and its hydrolysis back to the starting materialis 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
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Substrate cycles can serve two functions:
1) Amplification of metabolic signals.
2) To generate heat which is releasedduring 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 netflux of B by 380%.
Reciprocal regulation is exquisitely
sensitive!!!
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Gluconeogenesis and Glycolysis are Reciprocally Regulated
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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 PDHis inhibited by acetyl CoA. Thus excess acetyl CoAslows its formation from pyruvate and stimulates gluconeogenesis byactivating pyruvate carboxylase. 25Dr.Santosh Kumar
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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 lowconcentration during starvation and high concentration in the fed statedue to the antagonistic effects of glucagon and insulin on its production.
Second Coordinated Control Point:Fructose 1,6-Bisphosphate Fructose 6-phosphate
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The activities of PFK2 and FBPase2 reside on the same polypeptidechain.
Both activities are reciprocally regulated by phosphorylation of asingle 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!!!!
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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 promotinglipolysis in adipose tissue. This effect is antagonized by insulin.
The result is increased fatty acid oxidation by the liver whichpromotes 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, whichactivates protein kinase A, which then phosphorylates andINACTIVATES pyruvate kinasethereby decreasing glycolysis.
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Glucagon stimulates gluconeogenesis by decreasing the concentrationof F-2,6-BP in the liver.
Mechanism:Glucagon adenylate cylase to produce cAMP, which thena cAMP-dependent protein kinase to phosphorylate PFK2/fructosebisphosphatase 2. This phosphorylation the kinase andthe phosphatase activity. Both of which lead to a in the F-2,6-BPlevel.
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 thusan increase in gluconeogenesis).
Insulin causes the opposite effects.29Dr.Santosh Kumar
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3) Glucagon and insulin mediate long-term effects by inducing andrepressing the synthesis of key enzymes.
Glucagon induces the synthesis of:
PEP-carboxykinaseGluconeogenic fructose 1,6-bisphosphataseenzymes glucose 6-phosphatase
certain aminotransferases
Glucagon represses the synthesis of:
glucokinaseGlycolytic PFK1
enzymes pyruvate kinase
Insulin generally opposes these actions.
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Thus a high glucagon/insulin ratio in the blood:
i)increases the enzymatic capacity for gluconeognesis
ii) decreases the enzymatic capacity for glycolysis
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The Cori Cycle
Lactate and alanine, produced by skeletal muscle and RBCs are themajor 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
32Dr.Santosh Kumar