Lecture 04 Gluconeogenesis

Gluconeogenesis - Introduction

Gluconeogenesis: The synthesis of glucose from noncarbohydrateprecursors (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 bygluconeogenesis.

•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 reactionsof gluconeogenesis areexact reversals of glycolysis.

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.

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 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 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 reducingequivalents that willbe needed later.

MitochondrialMalate dehydrog.

Pyruvatetransporter

Malate/α-KGtransporter

Produced in muscleOr 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-ketoglutaratecarrier.

Step 5: In the cytosol, malate is reoxidized to oxaloacetate via cytosolic malate dehydrogenase with the production of cytosolicNADH.

L-malate + NAD+ oxaloacetate + NADH + H+



Pyruvatetransporter



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 investmentof 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).



Pyruvatetransporter



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 to proceed (i.e., NADH is needed at the glyceraldehyde 3-phosphatedehydrogenase step). 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 (via pyruvate 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.



Pyruvatetransporter


Produced in muscleor RBCs

PEP

Pyruvate

Glucose

Glucose 6-phosphate



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

ΔG˚’ = -3.9 kcal/mol

PEP

Pyruvate

Glucose

Glucose 6-phosphate



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

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 turnof the cycle. Can get net synthesis of glucose from citric acid cycleintermediates.

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 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 specialimportance as they are used to transport amino groups from a varietyof tissues to liver deaminated topyruvate 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

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 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

•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, whichactivates protein kinase A, which then phosphorylates andINACTIVATES pyruvate kinase thereby decreasing glycolysis.

•Glucagon stimulates gluconeogenesis by decreasing the concentrationof 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 andrepressing 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 themajor fuels for gluconeogenesis.

Pyruvate lactate alanine

LDH

Transaminase

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

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Lecture 04 Gluconeogenesis