The glycerol-3-phosphate shuttle is a mechanism used in skeletal muscle and the brain^[1] that regenerates NAD+ from NADH, a by-product of glycolysis. The NADH generated during glycolysis is found in the cytoplasm and must be transported into the mitochondria to enter the oxidative phosphorylation pathway. However, the inner mitochondrial membrane is impermeable to NADH and NAD+ and does not contain a transport system for these electron carriers. Either the glycerol-3-phosphate shuttle pathway or the malate-aspartate shuttle pathway, depending on the tissue of the organism, must be taken to transport electrons from cytoplasmic NADH into the mitochondria.^[2] The shuttle consists of the sequential activity of two proteins; Cytoplasmic glycerol-3-phosphate dehydrogenase (cGPD) transfers an electron pair from NADH to dihydroxyacetone phosphate (DHAP), forming glycerol-3-phosphate (G3P) and regenerating NAD+ needed to generate energy via glycolysis.^[3] The other protein, mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) catalyzes the oxidation of G3P by FAD, regenerating DHAP in the cytosol and forming FADH₂ in the mitochondrial matrix.^[4] In mammals, its activity in transporting reducing equivalents across the mitochondrial membrane is considered secondary to the malate-aspartate shuttle.

The glycerol phosphate shuttle was first characterized as a major route of mitochondrial hydride transport in the flight muscles of blow flies.^[5][6] It was initially believed that the system would be inactive in mammals due to the predominance of lactate dehydrogenase activity over Glycerol-3-phosphate dehydrogenase 1 (GPD1)^[5][7] until high GPD1 and GPD2 activity were demonstrated in mammalian brown adipose tissue and pancreatic ß-islets.^{[8][9][10][11]}

In this shuttle, the enzyme called cytoplasmic glycerol-3-phosphate dehydrogenase 1 (GPD1 or cGPD) converts dihydroxyacetone phosphate (2) to glycerol 3-phosphate (1) by oxidizing one molecule of NADH to NAD⁺ as in the following reaction:

Glycerol-3-phosphate is converted back to dihydroxyacetone phosphate by an inner membrane-bound mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2 or mGPD), this time reducing one molecule of enzyme-bound flavin adenine dinucleotide (FAD) to FADH₂. FADH₂ then reduces coenzyme Q (ubiquinone to ubiquinol) whose electrons enter into oxidative phosphorylation.^[12] This reaction is irreversible.^[13] These electrons bypass Complex I of the electron transport chain, making the glycerol-3-phosphate shuttle less energetically efficient compared to oxidation of NADH by Complex I.^[14]

• Malate-aspartate shuttle
• Mitochondrial shuttle