Gluconeogenesis Pathway: Definition, Steps, Substrates, Importance, Regulation.

The Gluconeogenesis process mainly occurs during the periods of starvation, fasting, low-carbohydrate diets, or intense exercise when the glucose level becomes low in blood and other tissues.

All animals, plants, and microorganisms require glucose to perform their metabolic activity. Glucose is the primary energy source of human brain and nervous system, as well as the erythrocytes, testes, renal medulla, and embryonic tissues.

Gluconeogenesis Definition

• Gluconeogenesis is a metabolic pathway through which convert the pyruvate or three- and four-carbon containing compounds into glucose.
• This process is also known as Neoglucogenesis. 
• All plants, animals, fungi, bacteria, and other microorganisms perform Gluconeogenesis pathway.
• In mammals, Gluconeogenesis occurs mainly in the liver, and to a lesser extent in renal cortex and in the epithelial cells that line the interior of the small intestine.
• After the formation of glucose, it passes into the blood to supply other tissues.
• It is a reverse process of the glycolytic pathway, where glucose is formed from pyruvate. These two processes are running in opposite directions, although they do share some steps.
• 7 of the 10 enzymatic reactions of gluconeogenesis are the contrary of glycolytic reactions. The remaining three reactions are irreversible.
• The three irreversible steps are the conversion of pyruvate to Phosphoenolpyruvate, Fructose 1,6 bisphosphate to Fructose 6 phosphate, and Glucose 6 phosphate to glucose.

Gluconeogenesis Pathway Steps

Gluconeogenesis may occur in the liver or kidney, in the mitochondria or cytoplasm of those cells. In this pathway, pyruvate is converted into Glusoe through a series of reactions catalyzed by different enzymes.

1. In the first step, the pyruvate is converted into Oxaloacetate with the formation of ATP to ADP. The reaction is catalyzed by the enzyme Pyruvate carboxylase. 
2. Pyruvate carboxylase was first discovered in 1960, by a scientist called Merton Utter. It is a mitochondrial enzyme. This enzyme assists the passage of pyruvate from the cytosol to the mitochondrial matrix with the help of MPC-1 and MPC-2 complexes.
3. The enzyme mitochondrial malate dehydrogenase performs the reduction of Oxaloacetate into malate within the mitochondria. Now the malate passes through the malate aspartate shuttle in the inner mitochondrial membrane with the help of malate α-ketoglutarate transporter and comes to the cytosol.
4. In cytosol, malate oxidized into oxaloacetate by the enzyme called cytosolic malate dehydrogenase.
5. The oxaloacetate is first decarboxylated and then phosphorylated to yield phosphoenolpyruvate in presence of Mg2+ and Mn2+ ions. The reaction is catalyzed by the enzyme PEP Carboxykinase. In this reaction, GTP is hydrolyzed to GDP. The enzyme Phosphoenolpyruvate (PEP) carboxykinase helps in the removal of carbon dioxide molecules from oxaloacetate.
6. Phosphoenolpyruvate carboxykinase is an isoenzyme and available in both mitochondria and cytosol.
7. The next steps are the same as reversed glycolysis. But for the formation of fructose 6 phosphate from fructose 1,6 bisphosphate one molecule of water is required with the release of one phosphate. The reaction is catalyzed by the enzyme fructose 1,6 bisphosphatase.
8. The enzyme fructose 1,6 bisphosphatase hydrolyzes the C-1 phosphate in the fructose 1, 6- bisphosphate without releasing any ATP.
9. Next, the glucose 6 phosphate is formed from the fructose 6 phosphate with the help of the enzyme phosphoglucoisomerase.
10. The glucose 6 phosphate is converted into glucose with the help of the enzyme glucose 6 phosphatase. Glucose 6 phosphatase requires Mg2+ ions to catalyze the reaction. This step of Gluconeogenesis occurs in liver, kidney.
11. After the formation of glucose, it shuttled into the cytoplasm via the glucose transporters of the endoplasmic reticulum.

Gluconeogenesis Reactions

1. [Pyruvate] +  [HCO3-] + [ATP]  → [oxaloacetate] + [ADP] + [Pi]
2. [Oxaloacetate] + [GTP] ⇌ [phosphoenolpyruvate] + [CO2] + [GDP]
3. [Phosphoenolpyruvate] + [H2O] ⇌ [2-phosphoglycerate]
4. 2-Phosphoglycerate  ⇌ 3-phosphoglycerate
5. 3-Phosphoglycerate + ATP  ⇌ 1,3-bisphosphoglycerate + ADP
6. 1,3-Bisphosphoglycerate + NADH + [H+]  ⇌ glyceraldehyde + 3-phosphate + NAD + Pi
7. Glyceraldehyde 3-phosphate ⇌ dihydroxyacetone phosphate
8. Glyceraldehyde 3-phosphate + dihydroxyacetone phosphate  ⇌ fructose 1,6-bisphosphate
9. Fructose 1,6-bisphosphate → fructose 6-phosphate + Pi
10. Fructose 6-phosphate ⇌  glucose 6-phosphate
11. Glucose 6-phosphate + H2O  → glucose + Pi

Final Reaction of Gluconeogenesis

2 Pyruvate + 4ATP + 2GTP + 2NADH + [2H+] + 4H2O → glucose + 4ADP + 2GDP + 6Pi + [2NAD+]

Importance of Gluconeogenesis 

1. Fulfill the energy demands of skeletal muscle, RBCs, neurons,  embryonic tissue, medulla of the kidney, testes, etc.
2. During starvation, it plays an important role in blood glucose homeostasis.

Substrate for Gluconeogenesis 

There are present different substrates for Gluconeogenesis such as Glycerol, lactate, and Glucogenic amino acids.

Glycerol

Glycerol is produced from the triglyceride hydrolysis in the adipose tissue. After that, it moves to the liver via the bloodstream.

Glycerol enters into the Gluconeogenesis pathway in two sequential steps:

1. In the first step, Glycerol is phosphorylated to form Glycerol 3 phosphate with the help of the enzyme Glycerol kinase. In this reaction, one ATP molecule is used.
2. After that, the Glycerol 3 phosphate is oxidized to form dihydroxyacetone phosphate. In this reaction, one molecule of NAD is reduced to NADH. The reaction is catalyzed by the enzyme Glycerol 3 phosphodehydrogenase.
3. It also clears the metabolites which are accumulated in blood such as lactate and glycerol etc.

Lactate

Lactate is produced via anaerobic glycolysis in RBC and muscles. After that, Lactate moves to the liver via the bloodstream.

In liver lactate converted into Pyruvate by the enzyme lactate dehydrogenase. Now Pyruvate enters into the Gluconeogenesis pathway and produces glucose.

Glucogenic amino acids

Glucogenic amino acids are produced by the hydrolysis of tissue proteins. Some examples of Glucogenic amino acids are Succinyl Co-A, α-ketoglutarate, fumarate, oxaloacetate, and fumarate.

Glucogenic amino acids enter into the Gluconeogenesis pathway via two entry points such as pyruvate and oxaloacetate.

Regulation of Gluconeogenesis 

Acetyl CoA

Acetyl CoA regulates the conversation of Pyruvate to Phophoenolpyruvate. It has both positive and negative regulation on Gluconeogenesis such as;

1. Positive: It increases the enzyme activity of pyruvate carboxylase as a result more oxaloacetate is produced.
2. Negative: It inhibits the pyruvate dehydrogenase as a result the conversation rate of pyruvate carboxylase to acetyl Co-A is decreased.

Glucagon

It is a hormone which is secreted from the α-cells of pancreatic islets. It regulates the conversation of fructose 1, 6-bisphosphate to fructose 6-phosphate. Glucagon regulates the Gluconeogenesis by two mechanisms such as; 

• It mediates the cyclic AMP, which inactivates the pyruvate kinase, as a result, it decreases the conversation rate ofPEP to pyruvate.
• It inhibits the phosphofructokinase and activates the fructose 1, 6-bisphosphate to increase the glucose synthesis.

Glucogenic amino acids

It regulates the conversion rate of glucose 6-phosphate to glucose during the time of decreased insulin level. 

References

1. https://biologyreader.com/gluconeogenesis.html
2. https://biologydictionary.net/gluconeogenesis/
3. https://en.wikipedia.org/wiki/Gluconeogenesis