Gluconeogenesis, which is the body's own synthesis of glucose, confronts some students who just studied glycolysis with the question: Why? Why  build up sugar again which has already been split up into its components? However, this metabolic pathway is more than just a reversal of glycolysis; in fact, it is essential to human life. Therefore, the following article will present you a comprehensive overview of  the processes of gluconeogenesis, so that you will be able to score with this extensive knowledge in your next biochemistry exam.
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Image: “The Glucose Response Curve” by openi. License: CC BY 2.0

Function of Gluconeogenesis

Gluconeogenesis provides the body with glucose when this cannot be obtained from food, such as during a fasting period. Some organs and cells, for instance, cannot gain energy from fat. Especially the red blood cells (erythrocytes), the renal medulla, and the nervous system depend on glucose as their main and sole energy source.

Location and Substrates of Gluconeogenesis

The enzymes necessary for gluconeogenesis are located in the human liver, the kidneys, and the intestinal mucosa. Here, the production of glucose from lactate (derived from muscle and erythrocytes), from glycogenic amino acids (mainly from the muscles) and from glycerol (especially from fat) is carried out. Lactate and glycogenic amino acids are converted into pyruvate and thus introduced to the pathway of gluconeogenesis.

Gluconeogenesis summary

Image: “gluconeogenesis summary” by Boumphreyfr. License: CC BY-SA 3.0

Glycerol is converted via two intermediate stages to fructose-1,6-bisphosphate, a metabolite of gluconeogenesis. Within the cell, some of the reactions have a mitochondrial course, which means that certain metabolites must be transported through the mitochondrial wall. The transport mechanisms will be discussed in more detail after the complete reaction.

The Steps of Gluconeogenesis

As gluconeogenesis is aimed at reversing glycolysis, the reversible steps of the glycolysis pathway simply run in the other direction. However, there are three irreversible steps that cannot run in the other direction for energetic reasons. These steps must be circumvented using three key reactions that make them more energy efficient.

Step 1: Conversion of Pyruvate to Phosphoenolpyruvate

Phosphoenolpyruvat formula

Image: “Struktur von Phosphoenolpyruvat in Fischer-Projektion” by NEUROtiker. License: Public Domain

Pyruvate is carboxylated by pyruvate carboxylase to oxaloacetate using 1 CO2 and 1 ATP. Oxaloacetate is decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase (PEPCK) to phosphoenolpyruvate by using 1 GTP and by releasing CO2. Here, decarboxylation drives the reaction; the phosphorylation generates a high-energy bond in the phosphoenolpyruvate. This energy comes from the GTP used.

Step 2 6: Conversion of Phosphoenolpyruvate to Fructose-1,6-Biphosphate

Fructose 16 bisphosphat formula

Image: “Struktur von beta-D-Fructose-1,6-bisposphat” by NEUROtiker. License: Public Domain

Steps 2 to 6 are presented here in a summarized form because they correspond precisely to the process of the glycolysis—just in the opposite direction. Via the intermediate products 2-phosphoglycerate, 3-phosphoglycerate, 1,3-bisphosphate and glyceraldehyde-3-phosphate, fructose-1,6-biphosphate is formed. In this process, 1 ATP and 1 NADH+H+ are consumed. The individual reaction steps can be found here. Glycolysis and gluconeogenesis both involve the same enzymes. It is merely the direction of the reaction which is different.

Step 7: Dephosphorylation of Fructose-1,6-Bisphosphate to Fructose-6-Phosphate

Fructose 6 phosphat formula

Image: “Struktur von beta-D-Fructose-6-phosphat” by NEUROtiker. License: Public Domain

The second key enzyme has to dephosphorylate fructose-1,6-biphosphate. In the gluconeogenesis, the enzyme fructose-1,6-bisphosphatase catalyzes the dephosphorylation of the substrate to fructose-6-phosphate, thereby consuming 1 H2O (in glycolysis, it is phosphofructokinase 1 which catalyzes the phosphorylation).

Step 8: Conversion of Fructose-6-Phosphate to Glucose-6-Phosphate

This step occurs without any use of energy in correspondence with glycolysis.

Step 9: Dephosphorylation of Glucose-6-Phosphate to Glucose

In the third and final key reaction, glucose-6-phosphate dephosphorylates to glucose consuming 1 H2O. This reaction is catalyzed by glucose-6-phosphatase and takes place in the endoplasmic reticulum.

Note: The key enzymes of gluconeogenesis are:

  1. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase (PEPCK)
  2. Fructose-1,6-bisphosphatase
  3. Glucose-6-phosphatase

These enzymes are found almost exclusively in the kidney, the liver, and the intestinal mucosa, which is why gluconeogenesis only takes place there.


Image: “Gluconeogenesis” by Phil Schatz. License: CC BY 4.0

Cell Compartments of Gluconeogenesis – Mitochondrial Shuttles

Other than in glycolysis, one of the enzymes of gluconeogenesis, the biotin- and ATP-dependent pyruvate carboxylase, is located inside the mitochondria.
Its substrate—pyruvate—is transported into the mitochondrial matrix through a carrier and is converted to oxaloacetate. Now, there are two possible paths for the oxaloacetate:

  1. Oxaloacetate is converted by a mitochondrial PEPCK and leaves the mitochondrion as phosphoenolpyruvate through an electroneutral transport.
  2. Malate-aspartate shuttle: Oxaloacetate is reduced to malate by the mitochondrial malate dehydrogenase, resulting in 1 NADH. The malate then enters the cytosol using an exchanger protein and is oxidized by the cytosolic malate dehydrogenase to oxaloacetate. This oxidizes NADH to NAD+. Therefore, it was also transported out of the mitochondrium and is now available for gluconeogenesis (consumed in step 5).

The second path probably represents the main path since only the cytosolic PEPCK can be regulated.

Energy Balance of Gluconeogenesis

With pyruvate as a starting substance, the synthesis of 1 molecule of glucose consumes 4 ATP, 2 GTP and 2 NADPH+H+. Considering the entirety of the process, a direct reversal of glycolytic pathway would be more efficient. However, as mentioned above, three steps of glycolysis have to be bypassed since with regard to thermodynamics, the required amount of energy would be too great.

With their circumvention, glucose synthesis becomes possible but is always endergonic. If this were not the case, the body could build and consume glucose in an endless cycle in order to produce energy. As it is, however, gluconeogenesis will only be used by the body when an insufficient glucose supply makes it absolutely necessary.

The Glucose Response Curve

Image: “The Glucose Response Curve” by openi. License: CC BY 2.0

Reactions of Gluconeogenesis

Reaction #1

  • Step 1 of bypassing pyruvate kinase
  • Occurs in mitochondrion

Reaction #2

  • Occurs in cytoplasm
  • Second triphosphate necessary

Reaction #9

  • Not the reversal of the PFK reaction
  • Energy realized by not regenerating ATP

Reaction #11

  • Not the reversal of the hexokinase reaction
  • Energy realized by not regenerating ATP
  • Occurs in endoplasmatic reticulum

Regulation of Gluconeogenesis

Since glycolysis and gluconeogenesis run in exactly opposite directions, it is important that they do not run simultaneously. Rate-controlling steps (effective within minutes) are:

Conversion of Pyruvate to Phosphoenolpyruvate

Pyruvate carboxylase catalyzes this first important step of gluconeogenesis. It is activated by acetyl-CoA so that more oxaloacetate is produced and available for further reaction steps.

Conversion of Fructose-1,6-Bisphosphate to Fructose-6-Phosphate

Fructose-1,6-bisphosphatase is responsible for this step. It is allosterically controlled by fructose-2,6-bisphosphate, just like its “opponent” – the phosphofructokinase. The regulatory enzyme is phosphofructokinase 2 (not to be confused with phosphofructokinase 1 of the glycolysis!).

When active, it synthesizes fructose-2,6-bisphosphate. It has an inhibitory effect on gluconeogenesis and a promoting effect on glycolysis. This kind of contrary regulation is also called reciprocal regulation. A stimulator of phosphofructokinase 2 is a low cAMP level in the cell, which, for example, is provided by insulin. Citrate and ATP have a positive effect on the new synthesis of glucose.

In the long term, the expression of key enzymes can be regulated. cAMP and glucocorticoids stimulate the expression whereas insulin represses the expression.

Review Questions

Solutions can be found below the source references.

1. What has a stimulating effect on gluconeogenesis?

  1. High insulin levels in the blood
  2. Activated phosphofructokinase 2
  3. ATP
  4. cAMP

2. Which cell compartment is not involved in gluconeogenesis?

  1. Liposome
  2. Endoplasmic reticulum
  3. Mitochondrion

3. In which organ or cells does gluconeogenesis take place?

  1. Brain
  2. Liver
  3. Erythrocytes
  4. Spleen
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