7 gluconeogenesis (5)

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