Copyright © 2000-2003 Mark Brandt, Ph.D.28GluconeogenesisIn animals, glucose is required by the brain, and is important to the properfunctioning of most tissues. A fall in plasma glucose can result in unconsciousness,and, if untreated, can be fatal. If dietary glucose is insufficient to maintain normalcirculating levels of glucose, additional glucose must be released from the liver. Theliver has some glucose stored in the form of glycogen but these stores only last forabout 12 hours in the absence of dietary glucose.Considerably before the glucose stores have been consumed, the organism mustbegin synthesizing additional glucose from other molecules in a process calledgluconeogenesis (literally “the birth of new glucose”). The vast majority ofgluconeogenesis occurs in the liver, with some additional glucose synthesisoccurring in the kidney.Glucose can be synthesized from pyruvate, from nearly all of the standard aminoacids, or from TCA cycle intermediates. Animals cannot convert acetyl-CoA toany of these compounds. Because the breakdown of fat results nearly exclusivelyin acetyl-CoA, fat (with minor exceptions) cannot be used to synthesize glucose.Instead, most glucose is synthesized either from lactate (produced during anaerobicglycolysis) or from amino acids (derived from breakdown of proteins).Some glucose is synthesized from glycerol (a breakdown product of triacylglyceroland phospholipids), and some can be synthesized from fatty acids containing an oddnumber of carbons. Cows and other ruminants use a breakdown product ofchlorophyll as a gluconeogenic substrate (in humans, this pathway is present but isfar less important). In general, however, gluconeogenesis uses amino acids eitherderived from dietary protein or from breakdown of proteins stores as the source ofthe substrate required for the process.Enzymes of the gluconeogenic pathwayMost of the enzymes used to synthesize glucose are also involved in the glycolyticpathway. Some glycolytic reactions, however, are irreversible under physiologicalconditions, and reversing these steps requires separate enzymes. The irreversiblesteps tend to act as regulatory control points.The diagram below summarizes the glycolytic and gluconeogenic pathways. Theenzymes shown in blue are the regulated glycolytic steps; the enzymes in red areregulated gluconeogenic enzymes. The enzymes shown in black are common to bothpathways.The irregular shape at the bottom of the diagram is a mitochondrion. Note that thepyruvate carboxylase reaction occurs exclusively in this compartment. The phospho-enolpyruvate carboxykinase reaction is shown in the cytoplasm; this is true forsome species; in humans the reaction occurs in both the cytoplasm (as shown) andin the mitochondria; in the latter case, the phosphoenolpyruvate is transported outof the mitochondria to allow the remainder of the gluconeogenic reactions toproceed.Copyright © 2000-2003 Mark Brandt, Ph.D.29Pyruvate carboxylaseThe pyruvate kinase reaction is physiologically irreversible. As a result, underphysiological conditions, converting pyruvate to phosphoenolpyruvate requires ashort pathway containing two important enzymes: pyruvate carboxylase andphosphoenolpyruvate carboxykinase. Pyruvate carboxylase is a mitochondrialenzyme that converts pyruvate to oxaloacetate. The pyruvate carboxylase reactionacts as both a mechanism for increasing the amount of TCA cycle intermediates(this function will be discussed further in the context of TCA cycle regulation) andas the first step of gluconeogenesis.Pyruvate carboxylase is a biotin-dependent enzyme; biotin is covalently bound tothe amino group of a pyruvate carboxylase lysine side-chain. Pyruvate carboxylasecatalyzes formation of a covalent between biotin and carbon dioxide (in the form ofCopyright © 2000-2003 Mark Brandt, Ph.D.30carbonate) in an ATP-dependent reaction; this carbonate is then transferred to thepyruvate substrate to produce oxaloacetate. (Because the ATP required for thisreaction is derived from catabolism of carbon compounds, the pyruvate carboxylasereaction does not involve net carbon fixation.)Phosphoenolpyruvate carboxykinasePhosphoenolpyruvate is an energetic molecule. Its production by phospho-enolpyruvate carboxykinase requires significant energy to form the high-energyphosphate bond in the molecule. The reaction is driven by GTP hydrolysis; inaddition, the reaction involves the loss of carbon dioxide, which acts as an entropicdriving force.Oxaloacetate cannot leave the mitochondria. This is potentially important,because the majority of the gluconeogenic enzymes are located in the cytoplasm. Inorder to allow gluconeogenesis to proceed, therefore, oxaloacetate must be convertedto a useful molecule that can be transported out of the mitochondria. Two moleculesfit this description: malate and phosphoenolpyruvate.In some animals (e.g., chickens and rabbits) the formation of phosphoenolpyruvateby phosphoenolpyruvate carboxykinase occurs in the mitochondria. In otheranimals, such as rats and mice, the phosphoenolpyruvate carboxykinase is locatedin the cytoplasm. In humans, phosphoenolpyruvate carboxykinase is found both inthe mitochondria and in the cytoplasm. Phosphoenolpyruvate produced in themitochondria can leave via a specific transporter.The alternative to mitochondrial phosphoenolpyruvate synthesis is to producemalate from oxaloacetate, because malate can be transported out of themitochondria. In this case, cytoplasmic malate dehydrogenase then reforms theoxaloacetate for conversion to phosphoenolpyruvate. The use of malate in thecytoplasm has advantages, because the conversion of malate to oxaloacetateproduces the NADH that will be required for the glyceraldehyde-3-phosphatedehydrogenase step. Thus, the use of malate transport effectively allows theCopyright © 2000-2003 Mark Brandt, Ph.D.31transfer of reducing equivalents from the mitochondrion to the cytoplasm. Inhumans, the location of phosphoenolpyruvate production is to some extentregulated by availability of NADH in the cytoplasm; if the cytoplasmic NADH levelis high, mitochondrial reducing equivalents are unnecessary for gluconeogenesis,and therefore phosphoenolpyruvate is produced in the