Gluconeogenesis: Pathway, Substrates, and Regulation

Gluconeogenesis

Overview

Gluconeogenesis is the metabolic pathway that synthesizes glucose from non-carbohydrate precursors, ensuring a continuous supply of glucose during periods of fasting or low carbohydrate intake. This process is essential for maintaining blood glucose levels, especially for tissues highly dependent on glucose, such as the brain and red blood cells.

• Glycogen stores in the liver can meet glucose needs for less than 24 hours in the absence of dietary carbohydrate intake.

• After prolonged fasting, hepatic glycogen stores are depleted, and glucose is synthesized de novo from non-carbohydrate sources.

• Gluconeogenesis requires both mitochondrial and cytosolic enzymes.

• During an overnight fast, approximately 90% of gluconeogenesis occurs in the liver, with the remaining 10% in the kidneys. During prolonged fasting, the kidneys contribute up to 40% of glucose production.

Substrates for Gluconeogenesis

Major Gluconeogenic Precursors

The primary substrates for gluconeogenesis are glycerol, lactate, and α-keto acids derived from glucogenic amino acids.

• Glycerol is released during the hydrolysis of triacylglycerols (TAG) in adipose tissue and transported to the liver.

• Glycerol is phosphorylated by glycerol kinase to form glycerol 3-phosphate, which is then oxidized by glycerol 3-phosphate dehydrogenase to dihydroxyacetone phosphate (DHAP).

• Lactate is released into the blood by exercising skeletal muscle and by cells lacking mitochondria (e.g., red blood cells).

• Through the Cori cycle, lactate is taken up by the liver, oxidized to pyruvate, and then converted to glucose, which is released back into the bloodstream.

• α-Keto acids are produced by the hydrolysis of tissue proteins and are major sources of glucose during fasting. Amino acid metabolism generates α-keto acids, such as pyruvate and oxaloacetate, which can enter the TCA cycle or be converted to phosphoenolpyruvate (PEP).

Key Reactions and Enzymes in Gluconeogenesis

Bypassing Irreversible Glycolytic Steps

Gluconeogenesis circumvents the three irreversible steps of glycolysis (catalyzed by hexokinase/glucokinase, phosphofructokinase-1, and pyruvate kinase) using four unique enzymes:

• Pyruvate carboxylase (PC)

• Phosphoenolpyruvate carboxykinase (PEPCK)

• Fructose 1,6-bisphosphatase

• Glucose 6-phosphatase

Pyruvate Carboxylation

The conversion of pyruvate to phosphoenolpyruvate (PEP) is a two-step process:

1. Pyruvate is carboxylated by pyruvate carboxylase (PC) to form oxaloacetate (OAA) in the mitochondria. This enzyme requires the coenzyme biotin, which is covalently bound to the ε-amino group of a lysine residue. The reaction involves the formation of an enzyme–biotin–carbon dioxide intermediate.

2. OAA is converted to PEP by phosphoenolpyruvate carboxykinase (PEPCK). This reaction is driven by the hydrolysis of GTP.

Pyruvate carboxylase is activated by acetyl CoA. In the absence of acetyl CoA, PC is largely inactive, and pyruvate is primarily oxidized by the pyruvate dehydrogenase complex or further oxidized by the TCA cycle.

Transport of Oxaloacetate to the Cytosol

• OAA cannot cross the mitochondrial membrane directly. It is reduced to malate by mitochondrial malate dehydrogenase, transported into the cytosol, and then reoxidized to OAA by cytosolic malate dehydrogenase. This process also transfers reducing equivalents (NADH) to the cytosol, which are required for gluconeogenesis.

Dephosphorylation Steps

• Fructose 1,6-bisphosphatase hydrolyzes fructose 1,6-bisphosphate to fructose 6-phosphate, bypassing the irreversible phosphofructokinase-1 (PFK-1) step of glycolysis.

• Glucose 6-phosphatase hydrolyzes glucose 6-phosphate to free glucose, bypassing the irreversible hexokinase/glucokinase step. This enzyme is present in the liver and kidneys but not in muscle or brain.

Regulation of Gluconeogenesis

Allosteric and Hormonal Regulation

• Fructose 1,6-bisphosphatase is inhibited by a high AMP/ATP ratio (indicating low energy) and by fructose 2,6-bisphosphate, an allosteric effector whose levels are regulated by the insulin/glucagon ratio.

• High ATP levels stimulate gluconeogenesis, while high AMP levels inhibit it.

• Acetyl CoA allosterically activates pyruvate carboxylase, linking fatty acid oxidation to increased gluconeogenesis during fasting.

• Glucagon increases cAMP levels, activating protein kinase A (PKA), which inactivates pyruvate kinase and favors gluconeogenesis over glycolysis. Glucagon also increases transcription of the PEPCK gene via cAMP response element-binding protein (CREB).

• Insulin has the opposite effect, decreasing gluconeogenic enzyme expression and activity.

Summary Table: Key Steps and Enzymes in Gluconeogenesis

Step	Enzyme	Location	Bypasses Glycolytic Enzyme
Pyruvate → Oxaloacetate	Pyruvate carboxylase	Mitochondria	Pyruvate kinase
Oxaloacetate → PEP	PEP carboxykinase (PEPCK)	Cytosol & Mitochondria	Pyruvate kinase
Fructose 1,6-bisphosphate → Fructose 6-phosphate	Fructose 1,6-bisphosphatase	Cytosol	Phosphofructokinase-1 (PFK-1)
Glucose 6-phosphate → Glucose	Glucose 6-phosphatase	Endoplasmic reticulum (liver, kidney)	Hexokinase/Glucokinase

Key Equations

• Overall reaction for gluconeogenesis (from 2 pyruvate): $2\ \text{Pyruvate} + 4\ \text{ATP} + 2\ \text{GTP} + 2\ \text{NADH} + 6\ \text{H}_2\text{O} \rightarrow 1\ \text{Glucose} + 4\ \text{ADP} + 2\ \text{GDP} + 6\ \text{P}_i + 2\ \text{NAD}^+ + 2\ \text{H}^+$

• Pyruvate carboxylase reaction: $\text{Pyruvate} + \text{CO}_2 + \text{ATP} + \text{H}_2\text{O} \rightarrow \text{Oxaloacetate} + \text{ADP} + \text{P}_i + 2\ \text{H}^+$

• PEP carboxykinase reaction: $\text{Oxaloacetate} + \text{GTP} \rightarrow \text{Phosphoenolpyruvate} + \text{GDP} + \text{CO}_2$

Clinical Relevance

• Deficiency of glucose 6-phosphatase (as in von Gierke disease) impairs gluconeogenesis and glycogenolysis, resulting in severe fasting hypoglycemia.

• Gluconeogenesis is critical during prolonged fasting, starvation, and intense exercise.

Additional info: Some explanations and context have been expanded for clarity and completeness based on standard biochemistry textbooks.