Gluconeogenesis

Gluconeogenesis

Word meaning: “formation of new sugar”

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

Gluconeogenesis is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as pyruvate, lactate, glycerol, and glucogenic amino acids.

Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms. The reactions are essentially the same in all tissues and all species.

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 takes place in the brain, skeletal muscle, or heart muscle.

Why do we synthesize glucose? 

  • Need to maintain glucose levels within a narrow range in blood.
  • Certain key organs, including the brain, can use only glucose as an energy source. Therefore, it is essential that the body maintain a minimum blood glucose concentration.
  • Brain, erythrocytes, testes & kidney medulla are dependent on glucose for continuous supply of energy. Hepatic glycogen can meet these needs for only 10 – 18 hours in the absence of dietary carbohydrate. Liver glycogen stores are depleted during a prolonged fasting.
  • Skeletal muscles in exertion (anaerobic conditions) use glucose at a rapid rate
  • Effectively prevent the accumulation of certain metabolites in blood. E.g. lactate, glycerol, propionate etc.

Precursors for gluconeogenesis:

  • The important precursors of glucose in animals are three-carbon compounds such as lactate, pyruvate, and glycerol, as well as certain amino acids, mainly alanine and glutamin (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
    (Citrate, isocitrate, alfa-ketoglutarate, succinyl coA, succinate, fumarate, malate etc all intermediates of TCA cycle are oxidized to oxaloacetate which then converts in glucose).
  • In many microorganisms, gluconeogenesis starts from simple organic compounds of two or three carbons, such as acetate, lactate, and propionate, in their growth medium.
  • Propionate, the principal glucogenic fatty acid produced in the digestion of carbohydrates by ruminants, is a major substrate for gluconeogenesis in these species.

Pathway

  • Gluconeogenesis is a pathway consisting of a series of eleven enzyme-catalyzed reactions. The pathway will begin in either the liver or kidney, in the mitochondria or cytoplasm of those cells, this being dependent on the substrate being used.
  • Gluconeogenesis and glycolysis are not identical pathways running in opposite directions, although they do share several steps. Seven of the ten enzymatic reactions of gluconeogenesis are the reverse of glycolytic reactions.
  • However, three reactions of glycolysis are essentially irreversible in vivo and cannot be used in gluconeogenesis:
  1. The conversion of glucose to glucose 6-phosphate by hexokinase
  2. The phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by phosphofructokinase-1
  3. The 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.
  • In gluconeogenesis, the three irreversible steps are bypassed by a separate set of enzymes, catalyzing reactions that are sufficiently exergonic to be effectively irreversible in the direction of glucose synthesis.
  • The enzymes for gluconeogenesis are located in the cytosol, except 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 share 7 reversible steps of glycolysis and the 3 irreversible steps are bypassed by the separate sets of enzymes.

Bypass step 1: Conversion of pyruvate to phosphoenolpyruvate

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)
Figure 1: Transport of PEP and oxaloacetate from the mitochondrion to the cytosol.

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

This final step in the generation of glucose does not take place in the cytosol. Rather, glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum, where it is hydrolyzed to glucose by glucose 6-phosphatase, which is bound to the membrane.

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

The generation of glucose is an important control point.

  • The fructose 6-phosphate generated by fructose 1,6-bisphosphatase is readily converted into glucose 6-phosphate. In most tissues, gluconeogenesis ends here. Free glucose is not generated; rather, the glucose 6-phosphate is processed in some other fashion, notably to form glycogen.
  • One advantage to ending gluconeogenesis at glucose 6-phosphate is that, unlike free glucose, the molecule cannot diffuse out of the cell.
  • To keep glucose inside the cell, the generation of free glucose is controlled in two ways.

1) The enzyme responsible for the conversion of glucose 6-phosphate into glucose, glucose 6-phosphatase, is regulated.

2) The enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis (tissues that release glucose into the blood). These tissues are the liver and to a lesser extent the 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.
  • Oxaloacetate is converted directly to PEP by a mitochondrial isozyme of PEP carboxykinase.
  • PEP is then transported across the mitochondrial membrane by specific membrane transport proteins to continue on the gluconeogenic path.
Figure 2: Alternative paths from pyruvate to phosphoenolpyruvate.

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 is phosphorylated to glycerol phosphate by glycerol kinase enzyme.
  • 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 phosphoenolpyruvate (an intermediate of glycolysis).
Figure 3: Involvement of citric acid cycle intermediates in gluconeogenesis

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. In addition, two molecules of NADH 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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