During a fast, amino acids produced by hydrolysis of tissue proteins are the main sources of glucose. The metabolism of these amino acids produces α-keto acids, such as pyruvate that can be converted to glucose, or α-ketoglutarate that can enter the tricarboxylic acid (TCA) cycle and form oxaloacetate (OAA), a direct precursor of phosphoenolpyruvate (PEP). However, acetyl coenzyme A (CoA) and compounds that only give rise to acetyl CoA cannot be used for the net synthesis of glucose. This is because the conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex (PDHC) is irreversible. These compounds instead give rise to ketone bodies and are known as ketogenic.

In the synthesis of glucose from lactate or pyruvate, seven glycolytic reactions are reversible, but three glycolytic reactions are irreversible. Therefore, four alternate reactions are necessary to energetically favor the synthesis of glucose. These four alternate reactions are unique to gluconeogenesis.

The first roadblock in the synthesis of glucose from pyruvate is the irreversible conversion of phosphoenolpyruvate (PEP) to pyruvate by pyruvate kinase (PK) in glycolysis. In gluconeogenesis, pyruvate is carboxylated by pyruvate carboxylase (PC) to oxaloacetate (OAA), which is then converted to PEP by PEP carboxykinase (PEPCK). Biotin is required as a coenzyme for PC, and the PC reaction occurs in the mitochondria of liver and kidney cells. It has two purposes: to produce PEP, an important substrate for gluconeogenesis, and to provide OAA to replenish the intermediates of the tricarboxylic acid (TCA) cycle. Muscle cells also contain PC, but they only use the OAA product for replenishment purposes and do not synthesize glucose.

PC is one of several carboxylases that rely on biotin as a coenzyme. Other carboxylases include acetyl CoA carboxylase, propionyl CoA carboxylase, and methylcrotonyl CoA carboxylase. The allosteric regulation of PC involves activation by acetyl CoA. Elevated levels of acetyl CoA in mitochondria indicate a metabolic state that requires increased synthesis of OAA. This occurs during fasting when OAA is used for gluconeogenesis in the liver and kidneys. Conversely, at low levels of acetyl CoA, PC is mostly inactive, and pyruvate is primarily oxidized by the PDHC to acetyl CoA, which can then be further oxidized by the TCA cycle.

After oxaloacetate is formed in the mitochondria, it needs to be transported to the cytosol for further steps in gluconeogenesis.
Amino acids produced by hydrolysis of tissue proteins are the major sources of glucose during a fast. Their metabolism generates α-keto acids, such as pyruvate that is converted to glucose, or α-ketoglutarate that can enter the tricarboxylic acid (TCA) cycle and form oxaloacetate (OAA), a direct precursor of phosphoenolpyruvate (PEP). [Note: Acetyl coenzyme A (CoA) and compounds that give rise only to acetyl CoA (for example, acetoacetate, lysine, and leucine) cannot give rise to a net synthesis of glucose. This is because of the irreversible nature of the pyruvate dehydrogenase complex (PDHC), which converts pyruvate to acetyl CoA (see p. 109). These compounds give rise instead to ketone bodies (see p.
195) and are termed ketogenic.]
III. REACTIONS
Seven glycolytic reactions are reversible and are used in the synthesis of glucose from lactate or pyruvate. However, three glycolytic reactions are irreversible and must be circumvented by four alternate reactions that energetically favor the synthesis of glucose. These irreversible reactions, which together are unique to gluconeogenesis, are described below.
A. Pyruvate carboxylation
The first roadblock to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of PEP to pyruvate by pyruvate kinase (PK). In gluconeogenesis, pyruvate is carboxylated by pyruvate carboxylase (PC) to OAA, which is converted to PEP by PEPcarboxykinase (PEPCK) (Fig. 10.3).
mitochondrial and cytosolic isozymes of malate dehydrogenase; GTP and GDP = guanosine tri-and diphosphates; ADP = adenosine diphosphate.
1. Biotin: PC requires the coenzyme biotin (see p. 385) covalently bound to the ε-amino group of a lysine residue in the enzyme (see Fig. 10.3). ATP hydrolysis drives formation of an enzyme–biotin–carbon dioxide (CO2) intermediate, which then carboxylates pyruvate to form OAA. [Note: HCO3− provides the CO2.] The PC reaction occurs in the mitochondria of liver and kidney cells and has two purposes: to allow production of PEP, an important substrate for gluconeogenesis, and to provide OAA that can replenish the TCA cycle intermediates that may become depleted. Muscle cells also contain PC but use the OAA product only for the replenishment (anaplerotic) purpose and do not synthesize glucose. [Note: Pyruvate carrier protein moves pyruvate from the cytosol into mitochondria.]
PC is one of several carboxylases that require biotin. Others include acetyl CoA carboxylase (p. 183), propionyl CoA carboxylase (p. 194), and methylcrotonyl CoA carboxylase (p. 266).
2. Allosteric regulation: PC is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA in mitochondria signal a metabolic state in which increased synthesis of OAA is required. For example, this occurs during fasting, when OAA is used for gluconeogenesis in the liver and kidneys. Conversely, at low levels of acetyl CoA, PC is largely inactive, and pyruvate is primarily oxidized by the PDHC to acetyl CoA that can be further oxidized by the TCA cycle (see p. 109).
B. Oxaloacetate transport to the cytosol