number 13

Done by Asma Karameh

Corrected by Saad hayek

Doctor Nayef Karadsheh

Gluconeogenesis

This lecture covers gluconeogenesis with aspects of:

1) Introduction to glucose distribution through tissues.

2) Definition of gluconeogenesis.

3) Precursors for gluconeogenesis.

4) Pathway of gluconeogenesis.

5) Regulation of gluconeogenesis.

6) Energy requirements for gluconeogenesis pathway.

Distribution of glucose: Free glucose in E.C.F 20grams which equals 80 calories.

Brain uses 120 g of glucose daily.

Liver stores up to hundred grams which forms 10% of the wet weight of the liver

Muscle stores up to 1%-2% of its weight.

Glycogen stored in the liver is about 75 grams, where in muscles it is about 400

grams of glycogen.

Liver glycogen maintains blood glucose for 16 hours.

A man with 70kg has 15 kg of fat which equals 130000kcal, an energy supply for

60-90 days.

ATP concentration at any moment is 15Mm

The concentrations of creatine phosphate is 20Mm

In post absorptive resting muscle or within moderate exercise, fatty acids are the

main source of energy, because the brain uses nearly 80% of glucose.

During prolonged fasting, ketone bodies serve as energy source as they are

produced from acetyl COA in B-oxidation of fatty acids.

Definition of gluconeogenesis:

It’s the process in which glucose is synthesized from non-carbohydrate

glucogenic precursors. It’s one of the mechanisms used to maintain blood

glucose levels. It occurs mainly in the liver under overnight fasting conditions

while 10% occur in the kidney, but under conditions of starvation, kidney

becomes the major glucose-producing organ. It produces 40% of glucose.

Notice:

1. Glucose synthesis doesn’t occur by a simply reversing the glycolysis; because the

overall equilibrium of glycolysis favors pyruvate formation.

2. Biomedically, Gluconeogenesis is also important in maintaining the levels of TCA

cycle intermediates, even when fatty acids are the main source of acetyl COA in

the tissues.

3. Gluconeogenesis is also useful in clearing glycerol produced by adipocytes, and

lactate produced by RBCs and muscles

Precursors of gluconeogenesis: Lactate :

It is mainly produced by exercising muscles, RBCs, cells that lack of mitochondria

and less vascularized cells. In fasting conditions, it’s converted to Pyruvate by a

dehydrogenation reaction which is reversible.

“Cori cycle" :- the produced lactate is taken up by the liver and oxidized to

pyruvate that is converted to glucose which is released back into circulation.

Amino acids:

- Those produced from hydrolysis of tissue proteins and can form pyruvate, are

the major sources of glucose during a fast. (Glucogenic amino acids).

- Alanine is the one mostly participates in gluconeogenesis.

- Metabolism of glucogenic amino acids produce alpha –keto acids.

- Ex. The reaction of converting Alanine to pyruvate:

1. Amino transferase transfers the amino group of alanine to alpha-keto gluterate

to form pyruvate and glutamate. The co-enzyme in this reaction is pyridoxal

phosphate which accepts and donates the amino group.

2. Then pyruvate forms oxaloacetate (OAA) which is a direct precursor of

phosphoenolpyruvate (PEP).

Notice:

1. Lysine and leucine are the only amino acids that can't participate in

gluconeogenesis, because they give rise only for acetyl Co-A which can't give rise

to net synthesis of glucose. Due to the irreversible reaction of pyruvate

dehydrogenase which converts pyruvate into acetyl Co-A. Thus instead Lysine

and Leucine are ketogenic amino acids producing ketone bodies and fatty acids.

2. Dr Nayef mentioned in his slides that alpha keto acids serve as precursors for

glucose synthesis.

Glycerol :

Glycerol is released from hydrolysis of triacylglycerol (TAG) in adipose tissue, and

is delivered to the liver by blood.

The carbons of glycerol are gluconeogenic because they form dihydroxyacetone

phosphate (DHAP) which is an intermediate of glycolysis and gluconeogenesis.

The pathway of generating DHAP: Glycerol is phosphorylated by glycerol kinase

to glycerol phosphate which is then oxidized by glycerol phosphate

dehydrogenase to Dihydroxyacetone phosphate (DHAP).

Propionate:

Fatty acids with an odd number of carbon atoms can form Propionyl-CoA from

the three carbons at omega end of the chain.

Propionyl CoA is then converted to methyl malonyl-COA and it is rearranged to

form succinyl CoA which can be used in gluconeogenesis.

The remaining carbons of the fatty acid goes under beta oxidation to form acetyl

CoA (which gives no rise to glucose synthesis).

Pathways of gluconeogenesis: - Gluconeogenesis differs from glycolysis because gluconeogenesis needs cytosolic

and mitochondrial enzymes. So it occurs in both compartments.

- - Starting with pyruvate, most of the steps of gluconeogenesis are the reverse of

those of glycolysis, but they differ in only three points which are regulated,

irreversible and constitute the energy barrier in glycolysis, so glycolysis or

gluconeogenesis occurs? Depends on the physiological conditions.

- 7 glycolytic steps are used in gluconeogenesis using the same enzymes that

catalyse the process of glycolysis. What differs is the flow of carbon atoms.

- However, the three irreversible glycolytic steps must be circumvented by four

alternate reactions that energetically favor the synthesis of glucose.

The unique reactions in gluconeogenesis:

1) Conversion of pyruvate to phosphoenolpyruvate (PEP):

-This reaction occurs in multiple steps.

- Pyruvate is first carboxylated by pyruvate carboxylase (PC) to oxaloacetate. In

this reaction, PC catalyzes the addition of CO2 to pyruvate .

- PC contains Biotin (vitamin B7) which is covalently bound to amino group of

lysine in the enzyme (this is the active form of the vitamin and it’s called biocytin).

- Biotin binds with Co2 and forms enzyme biotin-carbon dioxide intermediate.

-Then the Co2 Is transferred to pyruvate to form the carboxyl group of

oxaloacetate.

-To form the enzyme-biotin-CO2 intermediate, energy obtained from ATP

hydrolysis is required.

-this PC reaction which happens in the mitochondria has 2 purposes:

1- Allow production of PEP for gluconeogenesis which happens only in kidney and

liver cells

2-Replinish TCA cycle intermediate when depleted which is the only use of this

enzyme in muscle cells and this also happens in liver and kidney cells.

- OAA must be transported from the mitochondria to the cytosol. However the

OAA doesn’t readily cross the inner mitochondrial membrane and it doesn’t have

transporter to move it, so it’s converted to malate or Aspartate as they can

transvers the mitochondrial membranes (they work as carriers of oxaloacetate)

and the reduction of OAA to malate requires NADH.

-

-

-

-Reduction of oxaloacetate to malate is a reversible reaction requires energy in

the form of NADH and it occurs by mitochondrial malate dehydrogenase (MD).

-After malate enters the cytosol through its transporter, it’s oxidized to

oxaloacetate by cytosolic MD. In a reaction releases NADH. This oxaloacetate is

converted to PEP by cytosolic PEP carboxykinase (PEP-CK).

-The NADH produced is used to reduce 1,3-biphosphoglycerate to glyceraldehyde-

3-phosphate, which is a common step to both glycolysis and gluconeogenesis.

-OAA is decarboxylated and phosphorylated to PEP in the cytosol by PEP-CK, and

the reaction is driven by hydrolysis of GTP.

-OAA can be converted to PEP in the mitochondria by Mitochondrial PEP-CK, and

then PEP in transported to cytosol to complete gluconeogenesis, but this is not

the usual case. (The normal one is as explained earlier)

-PEP then undergoes the reactions of glycolysis running in the reverse direction

until it becomes fructose-1,6-bisphosphate

2) Conversion of phosphoenolpyruvate (PEP) to Fructose 1,6-

biphosphate:

-Starting with PEP as a substrate, the steps of glycolysis are reversed to form

glyceraldehyde-3-phosphate.

-For every two molecules of glyceraldehyde-3-phosphate that are formed, one is

converted to DHAP.

-The two triose phosphates DHAP and glyceraldehyde-3phosphate condense to

form fructose 1,6-biphosphate in a reaction that is the reverse of the aldolase

reaction.

-Fructose1.6-biphosohate is converted to fructose 6 phosphate by the irreversible

fructose 1,6- bisphosphatase 1 (FBP-1) reaction.

-Fructose-1,6-biphosphatase is an allosteric enzyme that participates in regulation

of gluconeogenesis.

Notice: glycerol participates at this level because it forms DHAP.

3) CONVERSION OF fructose 1,6-phosphate to fructose 6-phosphate:

-The enzyme fructose 1,6-biphosphatase 1(FBP-1) releases Pi from fructose1,6-

biphosphate to form fructose-6-phosphate. This isn’t a reversible reaction of

phosphofructokinase1 (PFK-1).

-The phosphate bond which is removed is a low energy phosphate bond, so no

ATP is formed during this reaction

-In the next step, fructose-6-phosphate is converted to glucose-6-phosphate by

phosphoglucoisomerase.

4) Conversion of glucose-6-phosphate to glucose.

-This is the last step in the generation of glucose.

-Glucose-6-phosphatase hydrolyzes Pi from glucose-6-phosphate and free glucose

is released into the blood. This isn’t a reversible reaction because the phosphate

bond is low energy bond and ATP isn’t generated in this step.

-This step is common to both glycogenlysis and gluconeogenesis.

-This reaction occurs by two steps, firstly glucose-6-phosphate must be trans-

located to the lumen of the endoplasmic reticulum where the enzyme glucose-6-

phosphatse is bound to the membrane at the luminal side.

-Therefore, this is a cytosolic step.

-This transfer occurs by glucose-6-phosphate translocase which moves inorganic

phosphate out as it transfers glucose-6-phosphate in.

-This step is the terminal step in both gluconeogenesis and glycogen degradation.

-Deficiencies in glucose-6-phasphatase leads to hypoglycemia in which glucose

can't be produced either by glycogenlysis or gluconeogenesis

Glucose and phosphate are then shuttled back to the cytosol by a pair of

transporters.

Energetics of gluconeogenesis:

-In gluconeogenesis, 6 moles of ATP are hydrolyzed in the synthesis of glucose,

Whereas two molecules of ATP are produced during glycolysis.

2 moles of ATP are used as two moles of pyruvate are carboxylated by PC.

2 moles of GTP are used to convert 2 moles of oxaloacetate into 2 moles of PEP.

2moles of ATP are used during phosphorylation of 2 moles of 3-phosphoglycerate

-Energy in the form of NADH is also required in the conversion of 1.3-

biphosphoglycerate into glyceraldehyde-3-phosphate

Notice: energy required for gluconeogenesis is obtained from Beta oxidation of

fatty acids (ATP and NADH)

So any defects in this process lead to hypoglycemia, because reduced fatty acid

derives energy production within the liver

If the product of gluconeogenesis which is glucose-6-phosphate enters glycolysis how many ATP

can be generated ?

The answer is 3 since glycolysis produces 4 ATP molecule but invest 2 in phosphorylation and

this glucose is already phosphorelated which will save one more ATP molecule.

Regulation of gluconeogenesis:

-Gluconeogenesis regulation is determined by two aspects:

1) Bioavailability of substrates:

-Gluconeogenesis is stimulated by the flow of it’s major substrates from

peripheral tissues into the liver.

-Glycerol and glucogenic amino acids are released from their sites in adipose and

muscles as levels of insulin are decreased while the levels of glucagon or stress

hormones like epinephrine and cortisol are elevated.

-In other words, the availability of gluconeogenic precursors strongly influences

the rate of glucose synthesis.

Activity or amount of key enzymes

-Two sequences in the pathway are regulated, as the net flow of carbons whether

in the glycolytic or gluconeogenic pathways depends on the activity of these

enzymes:

PyruvatePEP

Fructose1.6-biphosphatefructose-6-phosphate

1) Regulation of PDH & PC:

-Under fasting conditions, insulin levels are low, whereas glucagon levels are high.

-As a response, triacylglycerol and fatty acids are released from their stores in the

adipose tissue to be transferred to the liver with production of NADH, ATP, and

acetyl CoA which inactivates PDH, but activating pyruvate carboxylase.

- SO ACETYL COA works as an activator of PC (gluconeogenesis), and as an

inhibitor of PDH

2) Regulation of pyruvate phosphoenol carboxykinase:

This enzyme is an inducible one, in which the quantity of the enzyme in cells

increases by an increased transcription of its gene and increased translation to its

mRNA, and the main inducer is cAMP produced by glucagon whereas insulin

decreases its transcription.

GLUCAGON ACTIVATION OF cAMP activation of protein kinase A

Phosphorylation of specific transcription factorstranscription of PEPCK is

stimulatedincreased synthesis of PEPCK mRNA increased synthesis of the

enzyme.

Notice: cortisol is also involved in activating PEPCK but from a different site.

3) Regulation of pyruvate kinase:

-Elevated levels of glucagon also causes inhibition of this enzyme by

phosphorylating it using c-AMP dependant protein kinase A, so that PEP isn’t

reconverted to pyruvate.

4) Regulation of fructose1,6 biphosphatase:

Fructose 1,6-bisphosphatease (FBP-1) is inhibited by fructose 2,6-bisphosphate

(which is a very potent activator of phosphofructokinase 1 (PFK-1), THUS it

activates glycolysis and inhibits gluconeogenesis).

Fructose 2,6-bisphophate concentration is influenced by the insulin/glucagon

ratio, when glucagon is high, protein kinase A is activated and it phosphorylates

phosphofructokinase 2 (PFK-2) ,the enzyme that makes fructose 2,6-bisphosphate,

which will inactivate it and this will cause the activation of another subunit in this

same enzyme . which is fructose 2,6-bisphosphatase (FBP-2) which will

dephosphorylate the fructose 2,6-bisphosphate and convert it to fructose 6-

phosphate, thus reducing its concentration and inhibit glycolysis and activate

gluconeogenesis.

-Notice: In gluconeogenesis, the high levels of ATP also activates fructose1,6-

biphosphatase, while in glycolysis high levels of AMP activates

phosphofructokinase1. (Thus, elevated AMP stimulates energy-producing

pathways and inhibits energy-requiring ones).

*MA