Gluconeogenesis: Enzymes Involved, Steps, and Functions

During fasting, vigorous exercise, and hypoglycemic conditions, the body requires high glucose. Gluconeogenesis converts non-carbohydrate molecules like glycerol, pyruvate, lactate, glucogenic amino acids, and propionate to glucose molecules. 

Gluconeogenesis is essentially the reverse of glycolysis, a process that involves four key enzymes: pyruvate carboxylase, glucose-6-phosphatase, fructose-1,6-phosphatase, and phosphoenol pyruvate carboxykinase (PEPCK). These enzymes, operating in the three exergonic steps of glycolysis, are instrumental in the complex process of gluconeogenesis. 

Since glucose-6-phosphatase is not present in all human cell types, only specific cells can carry out this metabolic reaction. It occurs mostly in liver cells and, in some dire situations, in the renal cortex or kidney. 

Gluconeogenesis aids in supplying glucose during starvation. Glucose is the key fuel for most organs of the human body, including the brain. Although ketone bodies can be a secondary fuel source for the brain, other organs like the renal medulla, erythrocytes, and testes cannot survive without glucose.

Enzymes Involved in Gluconeogenesis

Many enzymes are involved in gluconeogenesis. It also requires reversible steps of glycolysis and enzymes involved in those. These include enolase, phosphoglycerate mutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, aldolase, and fructose-1,6-phosphatase. 

  1. Pyruvate carboxylase: This enzyme catalyzes the irreversible reaction of forming oxaloacetate from pyruvate. It is the first reaction in gluconeogenesis to reverse the irreversible reaction of glycolysis (phosphoenolpyruvate to pyruvate).  
  2. Malate dehydrogenase: It converts oxaloacetate to malate. Since this is a reversible reaction, the same enzyme also catalyzes the conversion of malate to oxaloacetate.  
  3. Phosphoenolpyruvate carboxykinase (PEPCK): This enzyme aids in phosphorylation and decarboxylation of oxaloacetate to form phosphoenolpyruvate. It reverses the irreversible reaction of glycolysis. 
  4. Enolase: It catalyzes the reversible reaction of converting phosphoenolpyruvate to 2-phosphoglycerate. 
  5. Phosphoglycerate mutase: It catalyzes the conversion of 2-phosphoglycerate to 3-phosphoglycerate. 
  6. Phosphoglycerate kinase: It phosphorylates 3-phosphoglycerate to form 1,3-biphosphoglycerate. 
  7. Glyceraldehyde-3-phosphate dehydrogenase: It converts 1,3-biphosphoglycerate to glyceraldehyde-3-phosphate. 
  8. Triose phosphate isomerase: It isomerizes glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. 
  9. Aldolase: It combines glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. 
  10. Fructose-1,6-phosphatase: This enzyme can dephosphorylate fructose-1,6-bisphosphate. The conversion of fructose-6-phosphate to fructose-1,6-phosphate in glycolysis is irreversible. So, this enzyme aids in reversing the nonequilibrium reaction.
  11. Glucose-6-phosphatase: This enzyme is present in smooth endoplasmic reticulum. It dephosphorylates glucose-6-phosphate to glucose.  

Steps of Gluconeogenesis

As discussed earlier, gluconeogenesis converts non-hexose compounds to glucose, a significant fuel for the body. So, the first step in gluconeogenesis is the conversion of these substrates to pyruvate. The pyruvate then converts into glucose via reversible glycolysis pathways. 

Substrate Conversion

Lactate or lactic acid is the product of fermentation in the liver, muscle, and red blood cells due to vigorous exercise. The enzyme lactate dehydrogenase is involved in the conversion of lactate to pyruvate. Here, a molecule of NADH+ and H+. 

During lipolysis, triglycerides break down into glycerol and fatty acids. Glycerol kinase then adds a phosphate molecule to the glycerol, forming glycerol-3-phosphate. This compound, glycerol-3-phosphate, plays a crucial role in our body’s energy metabolism. It can be oxidized to dihydroxyacetone phosphate, a key intermediate in glucose generation, by either the glycerol-3-phosphate dehydrogenase enzyme or the L- glycerol-3-phosphate oxidase enzyme. Dihydroxyacetone phosphate can then be reversed to generate glucose by reversing glycolysis.  

The breakdown of proteins generates amino acids. The amino acids can react with keto acid (alpha-ketoglutarate) to generate modified keto acid. Modified keto acid can convert to pyruvate, acetyl CoA, alpha-ketoglutaric acid, or succinyl CoA. These all can contribute to generating glucose. 

Pyruvate to Glucose

  1. Pyruvate first enters the mitochondria because the conversion of phosphoenol pyruvate to pyruvate is irreversible. Here, pyruvate carboxylase converts pyruvate to oxaloacetate instead of acetyl CoA. This reaction requires CO2, ATP (Adenosine Triphosphate), and coenzyme biotin. 
  2. Oxaloacetate cannot exit the mitochondrial membrane, so it is converted into malate through the reversible reaction of the Krebs cycle. The enzyme malate dehydrogenase catalyzes this reaction. Once the malate reaches cytosol, it converts back to oxaloacetate.
  3. Phosphoenolpyruvate carboxykinase (PEPCK) now decarboxylases oxaloacetate and rearranges to phosphoenolpyruvate. Hence, it reverses the nonequilibrium reaction of phosphoenolpyruvate. 
  4. Now, enolase converts phosphoenolpyruvate to 2-phosphoglycerate. 
  5. Phosphoglycerate mutase changes 2-phosphoglycerate to 3-phosphoglycerate. 
  6. Then, 3-phosphoglycerate is changed into 1,3-biphosphoglycerate by the enzyme phosphorylase kinase. Here, a molecule of ATP is utilized. 
  7. Now, the reversible reaction of glycolysis continues. Glyceraldehyde-3-phosphate dehydrogenase converts 1,3-biphosphoglycerate to glyceraldehyde-3-phosphate. Here, NADH (Reduced nicotinamide adenine dinucleotide) is the electron donor.
  8. After that, glyceraldehyde-3-phosphate isomerizes to dihydroxyacetone phosphate (DHAP) by the enzyme triose phosphate isomerase.
  9. Then, the enzyme aldolase catalyzes the combining of glyceraldehyde-3-phosphate and DHAP, forming fructose-1,6-biphosphate. 
  10. The dephosphorylation of fructose-1,6-biphosphate forms fructose-6-phosphate, catalyzed by the enzyme fructose-1,6-biphosphatase. This step reverses the nonequilibrium reaction by the enzyme phosphofructokinase. 
  11. Fructose-6-phosphate isomerizes to glucose-6-phosphate catalyzed by the enzyme phosphohexose isomerase. 
  12. Glucose-6-phosphate is dephosphorylated to glucose by the enzyme glucose-6-phosphatase in the smooth endoplasmic reticulum . This is the final reverse of the non-equilibrium reaction of glycolysis. 

Regulation of Gluconeogenesis

Gluconeogenesis is a crucial metabolic pathway for maintaining blood glucose levels during fasting or low-carbohydrate conditions. Its regulation is tightly controlled to ensure glucose homeostasis within the body. 

  1. Substrate availability: Gluconeogenesis is regulated by the availability of substrates such as lactate, glycerol, and certain amino acids. Increased levels of these precursors, especially lactate and amino acids like alanine, signal the need for glucose synthesis.
  2. Hormonal regulation:
    • Glucagon: Released by the pancreas in response to low blood glucose levels, glucagon stimulates gluconeogenesis by activating adenylate cyclase, increasing intracellular cyclic AMP (cAMP) levels. Elevated cAMP levels activate protein kinase A (PKA), leading to the phosphorylation and activation of enzymes involved in gluconeogenesis.
    • Cortisol: This stress hormone, released by the adrenal glands, also stimulates gluconeogenesis. Cortisol enhances the transcription of critical gluconeogenic enzymes and aids in the breakdown of proteins and fats to generate gluconeogenic precursors.
    • Insulin: In contrast to glucagon and cortisol, insulin inhibits gluconeogenesis. It decreases blood glucose levels by promoting glucose uptake in tissues and inhibiting glycogenolysis and gluconeogenesis in the liver. Insulin achieves this by suppressing the expression and activity of key gluconeogenic enzymes.
  3. Allosteric regulation of enzymes: Several enzymes involved in gluconeogenesis are subject to allosteric regulation, meaning their activity is controlled by binding specific molecules at sites other than their active sites. For example:
    • Phosphoenolpyruvate carboxykinase (PEPCK): This enzyme catalyzes an early step in gluconeogenesis. It is stimulated by glucagon and cortisol and inhibited by insulin.
    • Fructose-1,6-bisphosphatase (FBPase): It is inhibited by AMP but activated by citrate, an indicator of the cell’s high energy status.
    • Pyruvate carboxylase: Activated by acetyl-CoA, indicating an abundance of metabolic intermediates, and inhibited by ADP.

Functions of Gluconeogenesis

Gluconeogenesis has various functions in the body, mainly for maintaining blood glucose levels. It also provides energy during starvation, low-carbohydrate intake, and fasting. Brief functions of gluconeogenesis are as follows:

  1. Gluconeogenesis produces glucose during prolonged fasting or starvation. During longer durations of starvation and fasting, the regulation of bodily function can be adversely affected. Glucose is the main source of energy production for blood cells, brain cells, and the medulla of the kidney. In addition, gluconeogenesis also maintains blood glucose levels
  2. Besides producing glucose, gluconeogenesis also aids in conserving lean muscle mass. It spares muscle protein breakdown by providing an alternative source of glucose from non-carbohydrate molecules like amino acids. 
  3. Another vital function of gluconeogenesis is its role in energy metabolism. Glucose, released via gluconeogenesis, is a valuable resource for energy production through glycolysis. This process not only aids in energy production but also allows for the storage of glucose in the liver and muscles as glycogen for future use. 
  4. Gluconeogenesis supplies glucose to glycolytic tissues that heavily rely on glycolysis for energy production. However, these cells cannot produce glucose, so gluconeogenesis aids in providing glucose to such tissues. 
  5. Similarly, gluconeogenesis helps regulate blood pH by using H+ ions during some steps. This is important during conditions like diabetic ketoacidosis, where ketone bodies are produced in excessive amounts, which can lead to acidosis. 


  1. Chourpiliadis C, Mohiuddin SS. Biochemistry, Gluconeogenesis. [Updated 2023 Jun 5]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: 
  2. Melkonian EA, Asuka E, Schury MP. Physiology, Gluconeogenesis. [Updated 2023 Nov 13]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: 

Ashma Shrestha

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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