Gluconeogenesis

Word meaning: “formation of new sugar”

This process is frequently referred to as endogenous glucose production (EGP).

Gluconeogenesis definition

Gluconeogenesis can be defined as a metabolic pathway that leads to the formation of glucose from non-carbohydrate carbon compounds such as pyruvate, lactate, glycerol and glucogenic amino acids.

Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms.

Site: Occurs mainly in cytosol – some precursors are produced in mitochondria

In vertebrates, gluconeogenesis takes place mainly in the liver (Approx. 1 kg glucose/day) and to a lesser extent in the cortex of the kidneys.

About 90% of gluconeogenesis occurs in the liver and 10% in the kidney during an overnight fasting. However, during prolonged fasting, about 40% of gluconeogenesis occurs in the kidney.

Little gluconeogenesis occurs in the brain, skeletal muscle, or heart muscle.

Why do we synthesize glucose? 

  • Inorder to maintain glucose levels within a narrow range.
  • Some key organs, including the brain, can only use glucose as a source of energy. It is therefore essential for the body to maintain a minimum concentration of blood glucose.
  • For continuous energy supply, brain, erythrocytes, tests & kidney medulla are dependent on glucose. In the absence of dietary carbohydrate, hepatic glycogen can meet these needs for just 10 18 hours. During a prolonged fasting, liver glycogen stores are depleted.
  • Skeletal muscles in exertion (anaerobic conditions) use glucose at a rapid rate
  • The accumulation of certain metabolites in the blood is effectively prevented. Examples include lactate, glycerol, propionate, etc.

Precursors for gluconeogenesis:

  •  In animals  three-carbon compounds such as lactate, pyruvate, and glycerol, as well as certain amino acids, mainly alanine and glutamin act as important glucogenic precurssors.(Altogether, they account for over 90% of the overall gluconeogenesis)
  • Other substrates for gluconeogenesis include other glucogenic amino acids as well as all citric acid cycle intermediates
    ( Intermediates of TCA cycle including Citrate, isocitrate, alfa-ketoglutarate, succinyl coA, succinate, fumarate, malate  are oxidized to oxaloacetate and it is then converts to glucose).
  • The growth media for many microorganisms contain simple organic compounds like acetate, lactate, and propionate. They utilize these two or three carbon organic compounds as glycogenic precursor.
  • Ruminants produce propionate (glucogenic fatty acid) during the digestion of carbohydrates. In these species propionate act as a glucogenic precursor.

Gluconeogenesis Pathway

  • Eleven sequential enzyme catalysed reactions are there in gluconeogenesis.
  • The site where the pathway begins is depends on the precursor utilized (eg: In the mitochondria or cytoplasm of liver or kidney cells).
  • Even though gluconeogenesis and glycolysis have several common steps, they are not completely opposite pathways.
  • Among the ten enzymatic reactions in gluconeogenesis, seven of them are reverse of glycolytic reactions.
  • Three reactions of glycolysis are completely irreversible. They are:
  1. Hexokinase catalysed synthesis of glucose 6 phosphate from glucose
  2. PFK1 catalysed phosphorylation of fructose 6 phosphate to fructose 1, 6-bisphosphate
  3. Conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase
  • In cells, these three reactions are characterized by a large negative free-energy changeG), whereas other glycolytic reactions have a ΔG near 0.
  • The enzymes for gluconeogenesis are located in the cytosol.  Exception is seen for pyruvate carboxylase (in the mitochondria) and glucose 6-phosphatase (membrane bound in the endoplasmic reticulum).
  • Non carbohydrate precursors of glucose are first converted into pyruvate or enter the pathway in the form of oxaloacetate or dihydroxyacetone phosphate.

Steps in gluconeogenesis (When precursor is pyruvate)

Gluconeogenesis from pyruvate precurssor involves  7 reversible steps of glycolysis and the 3 irreversible steps. These steps are bypassed by different enzymes.

Bypass step 1: Phosphoenolpyruvate formation from pyruvate

The formation of phosphoenolpyruvate (PEP) from pyruvate (the reverse of the pyruvate kinase reaction) is endergonic and therefore requires free energy input. This is accomplished by first converting the pyruvate to oxaloacetate.

Oxaloacetate is a “high-energy” intermediate whose exergonic decarboxylation provides the free energy necessary for PEP synthesis.

The process (step 1) requires the participation of two enzymes

  1. Pyruvate carboxylase: Catalyzes the ATP-driven formation of oxaloacetate from pyruvate

Pyruvate carboxylase has a Biotin prosthetic group (functions as a CO2 carrier)

  1. PEP carboxy kinase (PEPCK): Converts oxaloacetate to PEP in a reaction that uses GTP as a phosphorylating agent.

Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves two endergonic reactions.

Endergonic reaction 1:Conversion of pyruvate to oxaloacetate (Carboxylation of pyruvate)

  • The enzyme for this reaction located in the mitochondria. So pyruvate is first transported from the cytosol into mitochondria (OAA can be is generated from glucogenic aminoacids within mitochondria by transamination)
  • Uses 1 molecule of ATP
  • Biotin binds CO2 from bicarbonate as carboxybiotin prior to the addition of the CO2 to pyruvate.

(In aqueous solutions, CO2 exists as HCO3- with the aid of carbonic anhydrase)

Endergonic reaction 2: Decarboxylation of oxaloacetate to PEP

  • Oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxy kinase in the cytosol.
  • This Mg 2+ dependent reaction requires GTP as the phosphoryl group donor (A molecule of GTP is hydrolyzed to GDP during this reaction).The overall equation for step 1 reaction:

(Two high-energy phosphate equivalents (one from ATP and one from GTP), each yielding about 50 kJ/mol under cellular conditions, must be expended to phosphorylate one molecule of pyruvate to PEP).

Gluconeogenesis requires metabolite transport between mitochondria and cytosol

  • Mitochondrial membrane has no transporter for oxaloacetate.
  • So before export to the cytosol the oxaloacetate formed from pyruvate must be converted either to aspartate (Route 1) or to malate (Route 2) for which mitochondrial transport systems exist.

The difference between these two routes involves the transport of NADH reducing equivalents

Route 2: The malate dehydrogenase route

  • Malate leaves the mitochondrion through a specific transporter in the inner mitochondrial membrane
  • This route results in the transport of reducing equivalents from the mitochondrion to the cytosol, since it utilizes mitochondrial NADH and produces cytosolic NADH.
  • Oxaloacetate is reduced to malate using NADH in the mitochondria
  • Malate is oxidized to oxaloacetate using NAD+ in the cytosol

 Route 1: The aspartate aminotransferase route

  • This route does not involve NADH. Cytosolic NADH is required for gluconeogenesis so, under most conditions, the route through malate is a necessity.
  • If the gluconeogenic precursor is lactate, its oxidation to pyruvate generates cytosolic NADH. So it can use route 1 or can be transported as PEP itself)

Bypass step 2: Conversion of Fructose-1, 6-bisphosphate to Fructose-6-phosphate (Exergonic hydrolysis)

Enzyme catalyzing the reaction: Mg2+ dependent Fructose 1,6- bisphosphatase (FBPase-1)

 Reaction:  Irreversible hydrolysis of the C-1 phosphate.

  • This reaction using one water molecule and releasing one phosphate
  • This is also the rate-limiting step of gluconeogenesis

 Bypass step 3: Conversion of Glucose-6-phosphate to glucose

Enzyme catalysing the reaction: Glucose-6-phosthatase

(Glucose-6-phosphatase is unique to liver and kidney, permitting them to supply glucose to other tissues. This enzyme is absent in muscle and adipose tissue.  Therefore they cannot export glucose into blood stream).

Reaction: Simple hydrolysis of a phosphate ester (Hydrolysis occurs at C6 of glucose 6 phosphate

Site of the reaction: Lumen of endoplasmic reticulum

Generation of glucose from glucose 6 phosphate does not take place in cytosol. Instead, glucose 6-phosphate is transported from cytosol to the lumen of the endoplasmic reticulum. ER membrane contain glucose 6-phosphatase enzyme, which will carry out the hydrolysis of glucose.

Glucose and Pi are then shuttled back to the cytosol by a pair of transporters.

The generation of glucose is an important control point.

  • In most of the tissues, gluconeogenesis ends with the formation of glucose 6-phosphate. They are not readily hydolysed to glucose. These glucose 6 phosphate is processed by some other pathway like glycogenesis.
  • You may wonder why it happens. This is because free glucose will readily diffuse out of the cell, where as glucose 6-phosphate molecule cannot diffuse out of the cell.
  • Two ways of controlling free glucose generation in cells:

1) Regulation of glucose 6-phosphatase enzyme (Catalyse the conversion of glucose 6-phosphate into glucose)

2) Limiting the presence of this enzyme to particular tissues that release glucose into blood especially liver and kidney

Gluconeogenesis when the precursor is lactate

Lactate is derived from anaerobic glycolysis in exercising skeletal muscle and in cells that lack mitochondria, and is delivered by the blood to the liver. Lactate is reconverted to pyruvate in the liver that forms glucose via gluconeogenesis (Cori cycle)

Conversion of pyruvate to PEP when the gluconeogenic precursor is lactate

When the gluconeogenic precursor is lactate, the scenario is different (no need of OAA to malate conversion)

Reason: The conversion of lactate to pyruvate in the cytosol of hepatocytes yields NADH, and the export of reducing equivalents (as malate) from mitochondria are therefore unnecessary.

  • After the production of pyruvate by the lactate dehydrogenase reaction, it is transported into the mitochondria.
  • In the mitochondria it is converted to oxaloacetate by pyruvate carboxylase, as described above.
  • Mitochondrial isozyme of PEP carboxykinase catalyse the direct  conversion of  Oxaloacetate  to PEP.
  • PEP is then transported across the mitochondrial membrane by specific membrane transport proteins to continue on the gluconeogenic path.

Gluconeogenesis when  the precursor is glycerol

  • Glycerol is formed from hydrolysis of triglycerides in adipose tissue, and is transferred to the liver via blood.
  • glycerol kinase enzyme catalyses the phosphorylation of glycerol to glycerol phosphate
  • Then glycerol phosphate is oxidized to dihydroxyacetone phosphate (an intermediate of glycolysis) by glycerol phosphate dehydrogenase enzyme

Gluconeogenesis when the precursor is glucogenic aminoacids

  •  Glucogenic amino acids are formed from the hydrolysis of proteins.
  • They are deaminated to α ketoacids such as α-ketoglutarate.
  • α-ketoglutarate is then converted to oxaloacetate via citric acid cycle or pyruvate (intermediate of glycolysis).
  • Oxaloacetate is a direct precursor of glycolysis pathway intermediate phosphoenolpyruvate (PEP)

Energetics of gluconeogenesis

In order for gluconeogenesis to generate one molecule of glucose, how many molecules of ATP and/or GTP are needed?

Answer:

For each molecule of glucose formed from pyruvate, six high-energy phosphate groups are required, four from ATP and two from GTP.

Apart from this, two  NADH molecules are required for the reduction of two molecules of 1,3-bisphosphoglycerate.

Explanation:

The sum of the biosynthetic reactions leading from pyruvate to free blood glucose is given below:

First bypass reaction: One molecule of ATP per molecule of pyruvate + 1 molecule of GTP per molecule of pyruvate

  • Enzyme pyruvate carboxylase converts pyruvate into oxaloacetate, which requires the input of one molecule of ATP per molecule of pyruvate used.
  • Enzyme PEP carboxykinase converts oxaloacetate into PEP, using one molecule of GTP per molecule of oxaloacetate used.

The conversion of 3-phosphoglycerate into 1,3-bisphosphoglycerate (1,3-BPG) by the enzyme phosphoglycerate kinase utilizes one molecule of ATP per molecule of 1,3-BPG generated. This is a reversible reaction. Now, we can add up the energy requirements.

Since each of these reactions needs to occur twice in order to generate a single molecule of glucose, we’ll need to multiply the energy investment by two in each step.

  • Thus, we have two molecules of ATP from the reaction catalyzed by pyruvate carboxylase.
  • We also have two molecules of GTP from the reaction catalyzed by PEP carboxykinase.
  • We have two molecules of ATP used from the reaction catalyzed by phosphoglycerate kinase.

Adding all of these up, we have a total of four molecules of ATP and two molecules of GTP.

(The conversion of lactate to pyruvate in the cytosol or the transport of reducing equivalents from mitochondria to the cytosol in the form of malate replaces the cytosolic NADH consumed in the glyceraldehyde 3-phosphate dehydrogenase reaction)

 

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