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Gluconeogenesis

Gluconeogenesis

5 min read Updated Apr 18, 2026

Gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors. It happens mainly in the liver and (to a lesser extent) the kidney. It is essentially glycolysis in reverse, but with four different “bypass” enzymes at the three irreversible steps of glycolysis.

Glycolysis vs gluconeogenesis: the four bypasses

Reciprocal pathways
Glycolysis ↓ Gluconeogenesis ↑ Glucose Glucose-6-P Fructose-6-P Fructose-1,6-bisP 2 × G3P / DHAP 2 × PEP 2 × Pyruvate phosphoglucose isomerase aldolase · triose-P isomerase 5 shared, reversible enzymes shared and reversible 1 hexokinase spends 1 ATP 2 PFK-1 spends 1 ATP · rate-limiting 3 pyruvate kinase makes 1 ATP glucose-6-phosphatase liver and kidney only · in the ER 4 fructose-1,6-bisphosphatase the rate-limiting step going up 3 Oxaloacetate pyruvate carboxylase in the matrix · biotin + ATP PEP carboxykinase spends GTP 1 2 Fuels that can climb back up Lactate Glucogenic amino acids Glycerol Fatty acids: never PDH runs one way only
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Irreversible in glycolysis Bypass enzyme (gluconeogenesis only) Junction metabolite

CostGluconeogenesis burns 4 ATP + 2 GTP + 2 NADH to make one glucose, while glycolysis returns only 2 ATP + 2 NADH. Running both at once would just heat the cell, which is why the two are reciprocally regulated.

Where it happensLiver mostly, kidney cortex in prolonged fasting. Only these tissues have glucose-6-phosphatase, so only these tissues can release free glucose into the blood.

What can feed itLactate (Cori cycle), glucogenic amino acids (mainly alanine), and glycerol from fat. Acetyl-CoA cannot: PDH is irreversible, so fatty acids can never make net glucose.

Gluconeogenesis is not glycolysis in reverse. Seven of the ten steps are shared, freely reversible enzymes. The other three are one-way, so four separate bypass enzymes are needed to get around them. Learn the four bypasses and you have learned the pathway.

Gluconeogenesis connects to the other metabolic pathways by running them backwards. The glycerol backbone from fat (from triglyceride breakdown) and amino acids (from muscle protein) both feed into gluconeogenesis at different points. Lactate from anaerobic glycolysis in muscle and RBCs is shipped to the liver (Cori cycle) and converted back to glucose here. Essentially, gluconeogenesis is the liver’s reverse-engineering machine that makes glucose when we cannot get it from food.

Why Gluconeogenesis Matters

During fasting, brain and red blood cells still need glucose. Once glycogen stores are depleted (~24 hours), the body must make glucose from precursors. Gluconeogenesis ensures a steady blood-glucose supply even during prolonged fasting.

Precursors

  • Lactate (from muscle / RBC glycolysis) → pyruvate → glucose. Cori cycle.
  • Glycerol (from triglyceride breakdown) → DHAP → glucose.
  • Glucogenic amino acids (especially alanine from muscle proteolysis) → pyruvate or TCA intermediates → glucose.

The Four Bypass Enzymes

Glycolysis has three irreversible steps (1, 3, 10). Gluconeogenesis cannot simply reverse them; it uses different enzymes to bypass.

  1. 1
    Pyruvate → OAA → PEP
    Pyruvate carboxylase (mito) + PEP carboxykinase · bypasses pyruvate kinase
    -1 ATP, -1 GTP
  2. 2
    F1,6BP → F6P
    Fructose-1,6-bisphosphatase · bypasses PFK-1 rate-limiting
    hydrolysis
  3. 3
    G6P → glucose
    Glucose-6-phosphatase · liver and kidney only · releases free glucose to blood
    hydrolysis

Total cost: 6 ATP equivalents per glucose made (vs. 2 ATP gained by glycolysis). Expensive - run only when glucose is truly needed.

Pyruvate to PEP

This two-step bypass of pyruvate kinase is the most energetically expensive part:

  1. Pyruvate carboxylase (in mitochondrion) adds CO2 to pyruvate, making oxaloacetate. Uses 1 ATP. Requires biotin (vitamin B7).
  2. PEP carboxykinase (PEPCK) decarboxylates oxaloacetate and phosphorylates it to PEP. Uses 1 GTP.

Pyruvate carboxylase is activated by acetyl-CoA - a signal that fat is being burned, so glucose is scarce, and the liver should synthesize more.

Glucose-6-Phosphatase

Only the liver and kidney have glucose-6-phosphatase. This enzyme removes the phosphate from G6P, producing free glucose that can leave the cell via GLUT2. Muscle lacks this enzyme - that is why muscle glycogen cannot directly contribute to blood glucose. Only liver glycogen can be broken down and released as blood glucose.

Cost

Per glucose synthesized from 2 pyruvate: 4 ATP + 2 GTP + 2 NADH consumed. Gluconeogenesis is expensive - the body only runs it when glucose is truly needed (fasting, low blood sugar, post-exercise).

What are the four bypass enzymes of gluconeogenesis, and which glycolysis steps do they bypass?
Click to reveal answer
Pyruvate carboxylase and PEP carboxykinase together bypass pyruvate kinase (step 10). Fructose-1,6-bisphosphatase bypasses PFK-1 (step 3). Glucose-6-phosphatase bypasses hexokinase (step 1). These four enzymes let the pathway run thermodynamically downhill in the opposite direction from glycolysis.
Why can humans not convert fatty acids to glucose?
Click to reveal answer
Beta-oxidation of fatty acids produces acetyl-CoA, not pyruvate. The pyruvate dehydrogenase reaction (pyruvate → acetyl-CoA) is irreversible - there is no enzyme to convert acetyl-CoA back to pyruvate. Without this step, fatty acid carbons cannot enter gluconeogenesis. The glycerol backbone of triglycerides CAN be converted to glucose; only the fatty acid tails cannot.
Why does muscle not contribute directly to blood glucose?
Click to reveal answer
Muscle lacks glucose-6-phosphatase, the enzyme that removes the phosphate from glucose-6-phosphate so glucose can leave the cell. Muscle glycogen breakdown produces G6P, which can be used locally (through glycolysis) but cannot be exported as free glucose. Only the liver and kidney have glucose-6-phosphatase and can directly contribute to blood glucose.
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