Gluconeogenesis is the synthesis of glucose from three carbon precursors such as alanine, pyruvate, lactate, and glycerol and is essentially a reversal of glycolysis  (Figure 13.3). Under normal conditions, 90% of gluconeogenesis occurs in liver, and the rest occurs mainly in the renal cortex. Other sites of gluconeogenesis, such as the small intestine have been suggested but remain controversial. Estimates of gluconeogenesis have been based on either arterio-venous differences across the splanchnic bed, magnetic resonance measurements of hepatic glycogen depletion during fasting, or a variety of isotope tracer methodologies. In humans, gluconeogenesis contributes 40 – 50% of endogenous glucose production after an overnight fast, though this number varies depending on the method used . During continued fasting, endogenous glucose production remains constant because gluconeogenesis rises in proportion to compensate for glycogen depletion. After about 48 hours of fasting in humans, all glucose is produced by gluconeogenesis. This is roughly the same timing at which people enter ketosis, a metabolic adaptation of liver to produce ketones from lipids and supplement glucose utilization in peripheral tissue. During extended fasting, total glucose production decreases as low insulin levels suppress peripheral glucose utilization, hepatic glycogen is depleted and the body adapts to ketone and lipid oxidation.
Most amino acids, organic acids and glycerol can be substrates for hepatic gluconeogenesis. Notably, liver lacks the enzymes to convert acetyl-CoA to glucose, thus even chain fatty acids, ketones and certain ketogenic amino acids cannot be used as gluconeogenic substrates, although mixing of carbon pools in the TCA cycle does occur  (Figure 13.3). Lactate and alanine are key components of the Cori cycle, the process by which glycolysis in peripheral tissue produces these intermediates and the circulation delivers them to the liver where they are converted to pyruvate and used to resynthesize glucose. Glycerol is produced by lipolysis in adipose tissue during fasting, and in humans contributes to about 10% of glucose production after an overnight fast, but increases during starvation and in diabetes [17,18]. Glycerol is distinct from other gluconeogenic precursors because it is technically already an alditol (e.g. sugar alcohol) and requires less free energy to be converted to glucose. Glycerol is converted to the gluconeogenic intermediate dihydroxyacetone phosphate by the combined actions of glycerol kinase and glycerol phosphate dehydrogenase, and thus bypasses several regulatory steps discussed later. All other gluconeogenic precursors must pass through mitochondrial pathways and their conversion to glucose is generally endergonic (Figure 13.3).
A minimum of 11 enzymatic steps are required to convert two molecules of pyruvate to one molecule of glucose . Seven of these enzymes catalyze reversible reactions of glycolysis such as aldolase and triose phosphate isomerase. Another four reactions are catalyzed by unique enzymes which circumvent irreversible steps of glycolysis (Figure 13.3). Gluconeogenesis from pyruvate begins with the transport of pyruvate into the mitochondria via the mitochondrial pyruvate carrier (MPC). Once in the mitochondria pyruvate is converted to oxaloacetate, by the mitochondrial enzyme pyruvate carboxylase (PC).
Oxaloacetate is then decarboxylated by phosphoenolpyruvate carboxykinase (PEPCK) to yield the glycolytic intermediate phosphoenolpyruvate (PEP). There are two isoforms of PEPCK, a cytosolic isoform (PEPCK-C) and a mitochondrial isoform (PEPCK-M). The human liver expresses about 50% of each, but the mouse liver expresses roughly 95% of its PEPCK as the cytosolic isoform . Studies in genetically engineered mice indicate that the cytosolic isoform is most important for gluconeogenesis .
Since mitochondria do not possess an oxaloacetate trans- porter, a modified malate-aspartate shuttle is utilized to transport substrates from mitochondria to the cytosol where PEPCK-C and other gluconeogenic enzymes are located. The exact form of this shuttle depends on cytosolic redox state. In its simplest form, mitochondrial oxaloacetate is reduced to malate by mitochondrial malate dehydrogenase with the oxidation NADH to NAD+ . Malate is transported out of the mitochondria in exchange for inorganic phosphate and then oxidized by cytosolic malate dehydrogenase to generate oxaloacetate and an NADH. Thus this pathway predominates when oxidized gluconeogenic substrates like pyruvate or alanine are utilized. Alternatively, mitochondrial oxaloacetate can be transaminated to aspartate and transported to the cytosol in exchange for glutamate, and then transaminated back to oxaloacetate. Although this process requires additional steps for transamination, it allows oxaloacetate to be transported in a redox neutral fashion (i.e., no net transport of NADH to the cytosol). The transaminase-dependent shuttle is critical for reduced substrates like lactate, but is not required for pyruvate itself.
Cytosolic phosphoenolpyruvate is converted to fructose-1,6- bisphosphate via six enzymatic steps common to glycolysis. However, conversion of fructose-1,6-bisphosphate to fructose-6-phosphate requires a distinct enzyme, fructose-1,6- bisphosphatase, as the ATP-consuming phosphofructokinase reaction of glycolysis is not reversible. Fructose-6-phosphate is then converted to glucose-6-phosphate by reversal of the hexose phosphate isomerase reaction of glycolysis. The terminal step of gluconeogenesis is the hydrolysis of glucose-6-phosphate (G6P) to free glucose, catalyzed by the glucose-6-phosphatase (G6Pase) enzyme complex. The complex is comprised of a catalytic subunit sequestered within the endoplasmic reticulum (ER), a glucose-6-phosphate translocase known as T1 that delivers glucose-6-phosphate to the catalytic subunit, and putative ER glucose and inorganic phosphate transporters (T2, T3) that move the reaction products back into the cytosol. The sequestration of G6Pase in the ER may protect other phosphorylated sugars from the nonspecific phosphohydrolase activity of its catalytic subunit. Specificity of the system is conferred by the T1 translocase component, which transports glucose-6-phosphate, but not its closely related epimer mannose-6-phosphate. This prevents uncontrolled hydrolysis of phosphorylated sugars and spares energy.