Gluconeogenesis (Compatibility Mode)

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 days requirement.
Dr.Santosh Kumar
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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.
Dr.Santosh Kumar
Entry of Noncarbohydrate Precursors

HK
Pyruvate
Glucose
PFK
Note places of entry of noncarbohydrate precursors.
Seven out of ten reactions of gluconeogenesis are exact reversals of glycolysis. Three steps in glycolysis are irreversible and thus cannot be used in gluconeogenesis. Therefore there are 3 steps for which bypass reactions are needed.
PK
Dr.Santosh Kumar
Glucose Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate
PEP
Pyruvate
Dr.Santosh Kumar
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.

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Malate/-KG transporter
Mitochondria are the source of reducing equivalents that will be needed later.
Mitochondrial Malate dehydrog.
Pyruvate transporter
Dr.Santosh Kumar
Produced in muscle Or RBCs
Step 3: Oxaloacetate is reduced to malate by mitochondrial malate dehydrogenase at the expense of mitochondrial NADH. Oxaloacetate + NADH + H+ L-malate + NAD+
Step 4: Malate exits the mitochondrion via the malate/-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+
Dr.Santosh Kumar
Dr.Santosh Kumar
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 + HCO3phosphoenolpyruvate + 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).
Dr.Santosh Kumar 9
Dr.Santosh Kumar
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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-phosphate dehydrogenase 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.
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Dr.Santosh Kumar
Produced in muscle or RBCs
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How do mito sense which pathway to follow? High cyt. lactate High cyt. NADH High cyt. malate
Mitochondrial Malate dehydrog. Malate/-KG transporter
High mit. malate High mit. OAA Shunts OAA into PEP production in mito.
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Produced in muscle or RBCs
PEP
Pyruvate
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Second Bypass Reaction: Conversion of Fructose 1,6bisphosphate 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 G = -3.9 kcal/mol fructose 6-phosphate + Pi
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PEP
Pyruvate
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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
G = -3.3 kcal/mol
glucose + Pi
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.*****
Dr.Santosh Kumar
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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.
Dr.Santosh Kumar
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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 -KG gluconeogenesis.
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Fatty Acids: even numbered carbon FA are not converted into glucose since during catabolism they yield only acetyl CoA which cant 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.
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Glycerol (which can be generated by hydrolysis of triacylglycerols (fat) to yield free FAs + glycerol) is an excellent substrate for gluconeogenesis.
Gluconeogenesis
Glycolysis
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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.
PFK1
ATP + fructose 6-phosphate Fructose 1,6-bisphosphate + H2O
ADP + fructose 1,6-bisphosphate + H+

Fructose 1,6-bisphosphatase
fructose 6-phosphate + Pi
Typically, both reactions are not simultaneously fully active in the same cell because of reciprocal regulation.
Dr.Santosh Kumar
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Substrate cycles can serve two functions: 1) Amplification of metabolic signals. Assume an allosteric effector: increases A decreases B B by 20% and A by 20%;
Result is to increase the net flux of B by 380%. Reciprocal regulation is exquisitely sensitive!!! 2) To generate heat which is released during the hydrolysis of ATP. ATP + H2O ADP + Pi + H+ Kumar Dr.Santosh + Heat
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Gluconeogenesis and Glycolysis are Reciprocally Regulated
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First Coordinated Control Point:
Pyruvate
PEP
(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.
(2) (1) (3)
(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.
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. Dr.Santosh Kumar 25
Second Coordinated Control Point: Fructose 1,6-Bisphosphate
Fructose 6-phosphate
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.
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PFK2
Fructose 6-phosphate
Fructose bisphosphatase 2
Fructose 2,6-bisphosphate
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 phosphatase. BOTTOM LINE: WHEN BLOOD GLUCOSE IS LOW: GLYCOLYSIS AND GLUCONEOGENESIS
27
F 2,6-BP, and then
PFK1 and
Fructose 1,6-bis-
SO THAT YOU MAKE MORE GLUCOSE IN THE LIVER!!!!

Dr.Santosh Kumar
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.
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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.
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3) Glucagon and insulin mediate long-term effects by inducing and repressing the synthesis of key enzymes. Glucagon induces the synthesis of: PEP-carboxykinase fructose 1,6-bisphosphatase glucose 6-phosphatase certain aminotransferases
Gluconeogenic enzymes
Glucagon represses the synthesis of: glucokinase PFK1 pyruvate kinase
Glycolytic enzymes
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

& RBC
Alanine Low NADH/NAD+ Ratio High NADH/NAD+ Ratio
Alanine
Lactate and alanine, produced by skeletal muscle and RBCs are the major fuels for gluconeogenesis. Pyruvate
LDH
lactate alanine
The cycle in which part of the metabolic burden is shifted from the muscle to the liver is known as the Cori Cycle.
Dr.Santosh Kumar 32

Gluconeogenesis (Compatibility Mode)

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Gluconeogenesis (Compatibility Mode)

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Gluconeogenesis - Introduction

During starvation or intense exercise, glucose must be replaced by gluconeogenesis.

Major site of gluconeogenesis: Liver

Secondary site: Kidney cortex.

Entry of Noncarbohydrate Precursors

Note places of entry of noncarbohydrate precursors.

Glucose Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate

First Bypass Reaction: Convervsion of Pyruvate to Phosphoenolpyruvate

Pyruvate carboxylase is a regulatory enzyme. Acetyl CoA is a positive effector.

Mitochondrial Malate dehydrog.

Produced in muscle Or RBCs

Step 4: Malate exits the mitochondrion via the malate/-ketoglutarate carrier.

Mitochondrial Malate dehydrog.

Produced in muscle Or RBCs

Mitochondrial Malate dehydrog.

Produced in muscle Or RBCs

Mitochondrial Malate dehydrog.

Produced in muscle or RBCs

Glucose Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate

Second Bypass Reaction: Conversion of Fructose 1,6bisphosphate to Fructose 6-phosphate

Glucose Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate

Third Bypass Reaction: Glucose 6-phosphate to Glucose

Gluconeogenesis from Various Metabolites

ATP + fructose 6-phosphate Fructose 1,6-bisphosphate + H2O

ADP + fructose 1,6-bisphosphate + H+

Gluconeogenesis and Glycolysis are Reciprocally Regulated

First Coordinated Control Point:

(1) Pyruvate Kinase: inhibited by ATP and alanine. Activated by F-1,6-BP.

(2) PEP Carboxykinase: ADP turns it off.

(2) (1) (3)

(3) Pyruvate Carboxylase: stimulated by acetyl CoA. Inhibited by ADP.

Second Coordinated Control Point: Fructose 1,6-Bisphosphate

F 2,6-BP, and then

SO THAT YOU MAKE MORE GLUCOSE IN THE LIVER!!!!

Hormonal Regulation of Gluconeogenesis

Glucagon represses the synthesis of: glucokinase PFK1 pyruvate kinase

Insulin generally opposes these actions.

Thus a high glucagon/insulin ratio in the blood:

The Cori Cycle

Alanine Low NADH/NAD+ Ratio High NADH/NAD+ Ratio

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