Welcome to part two of our lecture series on gluconeogenesis. This tutorial will cover the functions of the malate aspartate shuttle in the transport of oxaloacetate from the matrix of the mitochondria to the cytosol. In the last section, we discussed the pyruvate carboxylase enzyme reaction mechanism in the formation of oxaloacetate from pyruvate. This intermediate is required to generate phosphoenopyrruvate in a cytosolic reaction mediated by the phosphoenopyrruvate carboxykinase enzyme. Phosphoenopyrruvate carboxykinase, PEPCK, will mediate the removal of the carbon dioxide from oxaloacetate, followed by the phosphorylation of the intermediate to yield phosphoenopyrruvate. However, the transport of oxaloacetate from the matrix to the cytosol where it can be used as a substrate for this reaction is a problem. Oxaloacetate cannot cross the inner membrane of the mitochondria. To leave the mitochondrial matrix, oxaloacetate has to first be converted to a spartate by the spartate amino transferase enzyme AAT. It can then be transported to the intermembrane space of the mitochondria through an antiporter that transports one molecule of a spartate out of the matrix and one molecule of glutamate into the matrix. Once in the inner membrane space, the spartate can pass freely through a pore in the outer mitochondrial membrane. Once in the cytoplasm, a spartate is reconverted back to oxaloacetate using the cytoplasmic a spartate amino transferase enzyme. Oxaloacetate can then be used as a substrate by the phosphoenopyrruvate carboxykinase enzyme PEPCK. The glutamate a spartate transporter has some additional complexities associated with it. It cannot function on its own. It requires the coordinate functioning of the malate alpha-ketoglutarate antiporter. Together, these antiporters are known as the malate a spartate shuttle system. We will focus on the details of this system in the next few slides. The malate a spartate shuttle system is dependent on the functioning of two enzymatic processes. The first is the a spartate amino transferase that was indicated more simplistically on the previous two slides. The a spartate amino transferase enzyme can utilize glutamate as an amine donor to generate a spartate from oxaloacetate. Alpha-ketoglutarate is also formed in this process. Depending on substrate concentrations and other regulatory mechanisms, this enzyme can also work in the reverse direction to produce glutamate and oxaloacetate. In a different reaction using malate dehydrogenase, oxaloacetate can be reduced to form malate using a molecule of NADH as the electron donor. The malate dehydrogenase enzyme is expressed to high levels in the cytoplasm of liver cells, as well as the matrix of the mitochondria. This enzyme is a component of the crebs cycle, where it mediates the formation of oxaloacetate in the last step of the cycle. Within the cytoplasm, it predominantly converts oxaloacetate into malate. The malate can then be shuttled into the matrix of the mitochondria through the malate alpha-ketoglutarate anti-porter. In this anti-porter, malate moves into the mitochondrial matrix, while alpha-ketoglutarate moves into the inner membrane space, and subsequently into the cytozol of the cell. Together, with the a spartate, it can be utilized by cytoplasmic a spartate amino transferase to produce glutamate and oxaloacetate. This is the oxaloacetate pool that is then utilized in the gluconeogenic pathway. When PEPCK is active, the glutamate generated in this reaction is transported back into the matrix of the mitochondria through the a spartate glutamate anti-porter. The pool of a spartate in the matrix of the mitochondria is supplied by the reaction of the a spartate amino transferase enzyme, which completes the reverse reaction from the one seen in the cytoplasm. In the matrix, a spartate amino transferase uses glutamate and oxaloacetate, as substrates to generate a spartate and alpha-ketoglutarate, this enables the transport of a spartate and glutamate through their specific anti-porter. Within the gluconeogenic pathway, heightened levels of oxaloacetate are produced in the matrix of the mitochondria. Oxaloacetate is then converted to a spartate and transported across the inner membrane, where it can subsequently be converted back to oxaloacetate and used for the production of glucose.