Glycolysis and gluconeogenesis (Breakdown and synthesis of glucose)

Glycolysis versus gluconeogenesis

Glucose breakdown and synthesis are an essential process in the human body. Glucose provides the required substrates for aerobic and anaerobic metabolism. Glycolysis is the main route of metabolism for most carbohydrates (e.g., galactose, and fructose). Red blood cells, which lack mitochondria, even depend entirely on metabolizing glucose for energy and normal function. The metabolism of glucose is primarily controlled by hormones such as insulin and glucagon. Insulin is released in the postprandial state for anabolic metabolism, in which glucose is broken down to be transformed into storage forms (e.g., glycogen, fat). Conversely, glucagon predominates in the fasting state for catabolic metabolism, in which stored products are broken down (e.g., fats, amino acids) into glucose to be used as an energy source. The following table provides an overall comparison between glycolysis and gluconeogenesis.

Glycolysis Gluconeogenesis

Rate limiting enzyme

  • Glucagon
  • Stimulated in postprandial state
  • Occurs independent of postprandial state in some cells (e.g., erythrocytes)
  • Fasting state

Gluconeogenesis is more than just the reversal of glycolysis: The reactions of the key enzymes of glycolysis are irreversible due to thermodynamics and must therefore be reversed by different enzymes that are only active in gluconeogenesis.



  • Definition: A metabolic pathway that breaks down glucose by substrate-level phosphorylation and oxidation, yielding 2 pyruvate molecules and 2 ATP.
  • Location: cytosol of cells
  • Enzymes

Pyruvate kinase deficiency in erythrocytes causes chronic hemolytic anemia due to impaired glycolysis and a lack of ATP in the RBCs.

Sequence of reactions

Glucose is composed of a 6-carbon skeleton (C6H12O6). Each glucose molecule produces 2 pyruvate molecules, which are composed of a 3-carbon skeleton.

  1. Glucose → glucose 6-phosphate (G6P)
    • Enzyme
    • Requires ATP
  2. G6P → fructose 6-phosphate (F6P)
  3. F6P → fructose 1,6-biphosphate
    • Enzyme: PKF-1
    • Requires ATP
  4. Fructose 1,6-biphosphate → glyceraldehyde 3-phosphate (GAP)
    • Enzyme: aldolase
  5. GAP → 1,3-Biphosphoglycerate (1,3-BPG)
  6. 1,3-BPG → 3-phosphoglycerate
    • Enzyme: phosphoglycerate kinase
    • Produces ATP
  7. 3-phosphoglycerate → 2-phosphoglycerate
    • Enzyme: phosphoglycerate mutase
  8. 2-phosphoglycerate → phosphoenolpyruvate (PEP)
    • Enzyme: enolase
  9. PEP → pyruvate
    • Enzyme: pyruvate kinase
    • Produces ATP
    • Stimulated by fructose 1,6-biphosphate
    • Inhibited by ATP and alanine

The net reaction for glycolysis is as follows: glucose + 2 Pi + 2 ADP + 2 NAD+ → 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

In glycolysis, 2 ATP are being invested to gain 4 ATP, so in total, a net gain of 2 ATP per 1 molecule of glucose!

Glycolysis regulation

The regulation of glycolysis is determined by the activity of the enzymes hexokinase, phosphofructokinase-1, and pyruvate kinase, which catalyze essentially irreversible reactions in glycolysis and are therefore the main sites of control.

Enzyme Hexokinase Phosphofructokinase-1 Pyruvate kinase
  • Converts glucose to G6P
  • Converts F6P to fructose 1,6-biphosphate
  • AMP (indicates that energy is required → glycolysis is activated)
  • Fructose 2,6-bisphosphate
  • Insulin (in postprandial state): indirect stimulation by increasing levels of fructose 2,6-bisphosphate (see detailed mechanism below table)
  • Fructose-1,6-bisphosphate (feed-forward stimulation)
  • G6P (feedback inhibition)
  • Glucagon
  • ATP (indicates that energy is plentiful → glycolysis is slowed down)
  • Citrate (indicates plentiful supply of intermediates for energy production)
  • Low pH
  • Only in the liver: glucagon indirectly inhibits glycolysis by lowering levels of fructose 2,6-bisphosphate during a fasting state (see detailed mechanism below table)

Fructose-2,6-bisphosphate (feed-forward regulation)

Pyruvate metabolism

Pyruvate characteristics

LDH is found in almost every cell of the body. Elevated LDH levels without exercise may indicate cell injury due to cancer (e.g., germ cell tumors), hemolytic anemia, myocardial infarction, infection, kidney, or liver disease.

Products of pyruvate metabolism

Product Reaction Location Function Regulation
  • Pyruvatelactate (reversible)
    • Enzyme: lactate dehydrogenase (LDH)
      • Present in the heart, RBCs, and muscle, among others
      • Only occurs in anaerobic glycolysis
    • NADH is oxidized to NAD+ in the process
Acetyl-CoA Mitochondrion
Oxaloacetate Mitochondrion
Alanine Cytosol of myocytes

The five cofactors of the pyruvate dehydrogenase complex: Tender (Thiamine) Loving (lipoic acid) Care (CoA) For (FAD) Nancy (NAD+).

Arsenic inhibits lipoic acid, thereby preventing the production of acetyl-CoA and inhibiting the TCA cycle!

Pyruvate dehydrogenase complex deficiency results in impaired conversion of pyruvate to acetyl-CoA, a reduced production of citrate, and thus an impaired TCA cycle, leading to severe energy deficits (especially in the CNS). Long-term treatment includes a ketogenic diet (high fat, low carbohydrate, glucogenic amino acids, e.g. valine) and cofactor supplementation with thiamine and lipoic acid.




Primary substrates

All amino acids, except for leucine and lysine, can be used as substrates for gluconeogenesis.

Gluconeogenesis reactions and regulation

  • Gluconeogenesis is inhibited when there is an excess of energy available (i.e., large ATP/AMP ration) and activated if energy is required (i.e., low ATP/AMP ratio).
  • Gluconeogenesis is also stimulated by glucagon and inhibited by insulin (see phosphofructokinase-2 for the mechanism).
  • The following shows the key steps of gluconeogenesis that are irreversible and need to be bypassed with special enzymes. The other steps are merely the opposite reactions of glycolysis that are carried out by bidirectional enzymes (see glycolysis and this illustration ).
Enzyme 1. Pyruvate carboxylase → 2. Phosphoenolpyruvate carboxykinase → 3. Fructose-1,6-bisphosphatase → 4. Glucose-6-phosphatase
  • Converts fructose-1,6-bisphosphate to F6P
  • Converts G6P to glucose
Stimulated by:
  • Citrate
  • ATP
  • Fructose-1,6-bisphosphate (feed-forward regulation)
  • G6P (feed-forward regulation)
Inhibited by:
  • ADP
  • Fructose-2,6-bisphosphate
  • AMP

Clinical significance


Pentose phosphate pathway


In the pentose phosphate pathway, no ATP is produced or used up.

G6PD deficiency is the most common human enzyme deficiency. It results in insufficient NADPH production, which is required for reduction of the antioxidant glutathione to prevent excess hydrogen peroxide and free radicals from damaging RBC membrane (and causing hemolytic anemia).

Sequence of reactions (two phases)

Oxidative phase (irreversible)

Net reaction in 3 steps: G6P + 2 NADP+ + H2O → ribulose 5-phosphate + 2 NADPH + 2 H+ + CO2

  1. G6P6-phosphogluconolactone
  2. 6-phosphogluconolactone → 6-phosphogluconate
    • Enzyme: 6-phosphogluconolactonase
    • Requires 1 H2O
  3. 6-phosphogluconate → ribulose 5–phosphate

Nonoxidative phase (reversible)

Net reaction: 3 ribulose 5-phosphateribose 5-phosphate + 2 xylulose 5-phosphate ⇄ 2 fructose 6-phosphate + glyceraldehyde 3-phosphate

  • Function depends on the cell's needs:
  • Main reactions include:
    • Ribulose 5-phosphateribose 5-phosphate (isomerization reaction)
    • Ribulose 5-phosphate ⇄ xylulose 5-phosphate (epimerization reaction)
      • Enzyme: ribulose-5-phosphate epimerase (phosphopentose epimerase)
    • Ribose 5-phosphate + xylulose 5-phosphate ⇄ fructose 6-phosphate + glyceraldehyde 3-phosphate
  • 1. Anemaet IG, González JD, Salgado MC, et al. Transactivation of cytosolic alanine aminotransferase gene promoter by p300 and c-Myb. J Mol Endocrinol. 2010; 45(3): pp. 119–132. doi: 10.1677/jme-10-0022.
  • 2. van Schaftingen E, Gerin I. The glucose-6-phosphatase system. Biochem J. 2002; 362(Pt 3): pp. 513–32. pmid: 11879177.
last updated 01/06/2020
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