Gluconeogenesis requires the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) by the enzyme PEPCK. However, there is no transport system for OAA in the inner mitochondrial membrane. Instead, OAA is first reduced to malate by mitochondrial malate dehydrogenase (MD). Malate is then transported to the cytosol where it is reoxidized back to OAA by cytosolic MD, while nicotinamide adenine dinucleotide (NAD+) is reduced to NADH. The NADH is used in the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate by glyceraldehyde 3-phosphate dehydrogenase, a reaction that is common to both glycolysis and gluconeogenesis.

In situations where lactate is abundant, it is oxidized to pyruvate with the reduction of NAD+. The pyruvate is then transported into the mitochondria where it is carboxylated by pyruvate carboxylase (PC) to form OAA. This OAA can be further converted to PEP by the mitochondrial isozyme of PEPCK and transported to the cytosol. Alternatively, OAA can be converted to aspartate, which is transported into the cytosol.

The decarboxylation and phosphorylation of OAA to PEP in the cytosol is driven by the hydrolysis of guanosine triphosphate (GTP). This reaction is carried out by the enzyme PEPCK and provides an energetically favorable pathway from pyruvate to PEP. PEP is then subjected to the reverse reactions of glycolysis until it is converted to fructose 1,6-bisphosphate.

Fructose 1,6-bisphosphate can be hydrolyzed by fructose 1,6-bisphosphatase, which is found in the liver and kidneys. This bypasses the irreversible reaction catalyzed by phosphofructokinase-1 (PFK-1) in glycolysis and allows for the formation of fructose 6-phosphate through an energetically favorable pathway. This reaction is a critical regulatory step in gluconeogenesis.

The regulation of fructose 1,6-bisphosphatase is influenced by intracellular energy levels and fructose 2,6-bisphosphate. An increase in the AMP/ATP ratio inhibits fructose 1,6-bisphosphatase, indicating a low-energy state in the cell. Conversely, low AMP and high ATP levels stimulate gluconeogenesis. Fructose 1,6-bisphosphatase is also inhibited by fructose 2,6-bisphosphate, which is controlled by the insulin/glucagon ratio. In the presence of high glucagon levels, hepatic PFK-2 does not produce fructose 2,6-bisphosphate, resulting in the activation of fructose 1,6-bisphosphatase.

The final step of gluconeogenesis involves the dephosphorylation of glucose 6-phosphate.
For gluconeogenesis to continue, OAA must be converted to PEP by PEPCK. PEP production in the cytosol requires transport of OAA out of mitochondria. However, there is no OAA transporter in the inner mitochondrial membrane, and OAA is first reduced to malate by mitochondrial malate dehydrogenase (MD). Malate is transported into the cytosol and reoxidized to OAA by cytosolic MD as nicotinamide adenine dinucleotide (NAD+) is reduced to NADH (see Fig. 10.3). The NADH is used in the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3phosphate by glyceraldehyde 3-phosphate dehydrogenase (see p. 101), a reaction common to glycolysis and gluconeogenesis. [Note: When abundant, lactate is oxidized to pyruvate as NAD+ is reduced. The pyruvate is transported into mitochondria and carboxylated by PC to OAA, which can be converted to PEP by the mitochondrial isozyme of PEPCK. PEP is transported to the cytosol. OAA can also be converted to aspartate that is transported into the cytosol.]
C. Cytosolic oxaloacetate decarboxylation
OAA is decarboxylated and phosphorylated to PEP in the cytosol by PEPCK. The reaction is driven by hydrolysis of guanosine triphosphate ([GTP] see Fig. 10.3). The combined actions of PC and PEPCK provide an energetically favorable pathway from pyruvate to PEP. PEP is then acted on by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate.
The pairing of carboxylation with decarboxylation drives reactions that would otherwise be energetically unfavorable. This strategy is also used in fatty acid (FA) synthesis (see p. 184).
D. Fructose 1,6-bisphosphate dephosphorylation
Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase, found in the liver and kidneys, bypasses the irreversible phosphofructokinase-1 (PFK-1) reaction of glycolysis and provides an energetically favorable pathway for the formation of fructose 6-phosphate (Fig. 10.4). This reaction is an important regulatory site of gluconeogenesis.
1.
Regulation by intracellular energy levels: Fructose 1,6-bisphosphatase is inhibited by a rise in the adenosine monophosphate (AMP)/ATP ratio, which signals a low-energy state in the cell. Conversely, low AMP and high ATP levels stimulate gluconeogenesis, an energy-requiring pathway.
2.
Regulation by fructose 2,6-bisphosphate: Fructose 1,6-bisphosphatase is inhibited by fructose 2,6-bisphosphate, an allosteric effector whose concentration is influenced by the insulin/glucagon ratio. When glucagon is high, the effector is not made by hepatic PFK-2 (see p. 99), and thus, the phosphatase is active (Fig. 10.5). [Note: The signals that inhibit (low energy, high fructose 2,6-bisphosphate) or activate (high energy, low fructose 2,6-bisphosphate) gluconeogenesis have the opposite effect on glycolysis, providing reciprocal control of the pathways that synthesize and oxidize glucose (see p. 100).]
E. Glucose 6-phosphate dephosphorylation