Anaplerotic molecules: Current and future

The concept of anaplerosis
The oxidation of acetyl groups in the citric acid cycle (CAC) involves eight reactions, which (i) convert the two carbons of acetyl to CO2, and (ii) regenerate the acceptor of the acetyl group, i.e., oxaloacetate. When the only source of carbon entering the CAC is acetyl-CoA, the net fluxes through the eight reactions of the cycle are identical, although most of the reactions are reversible in intact cells (except citrate synthase and α-ketoglutarate dehydrogenase). The size of the total pool of the eight CAC intermediates (1–2 μmol/g) is small compared to the throughput of the cycle (1–2 μmol acetyl/g per min). This is why the eight intermediates are referred to as ‘catalytic intermediates’ of the CAC. Figure 1 illustrates the large differences between the sizes of the pools of individual intermediates. As a consequence, the turnover of these pools varies greatly: 5–10 times/min for citrate, and 100–200 times/min for oxaloacetate.

Although the reactions of the CAC provide 100% recovery of the catalytic intermediates, there is a physiological ‘leakage’ of intermediates through the mitochondrial membranes and the cell membranes, often referred to as ‘cataplerosis’. Although the word ‘cataplerosis’ is, sensu stricto, a misnomer, it is used extensively in the recent literature. The rate of physiological cataplerosis in the normal heart is estimated at 1–2% of the total pool per min. If the leakage of catalytic intermediates were not balanced by the re-filling reactions of anaplerosis, the flux through the CAC and the regeneration of ATP could not be sustained. Therefore, the maintenance of adequate pools of CAC intermediates is a conditio sine qua non of cell survival and homeostasis. Indeed, the mechanical performance of isolated rat hearts decreases rapidly when the perfusate contains only precursors of acetyl-CoA, i.e., acetate or acetoacetate. Recovery of cardiac mechanical performance follows the addition of an anaplerotic substrate (pyruvate, propionylcarnitine) to the perfusate.

Pyruvate is anaplerotic via pyruvate carboxylase and/or malic enzyme. Glutamate, and its abundant precursor glutamine, are converted to α-ketoglutarate by reactions catalysed by glutamate dehydrogenase and/or aminotransferases. Numerous precursors of propionyl-CoA (odd-chain fatty acids, propionylcarnitine, C5-ketone bodies) form succinyl-CoA via methylmalonyl-CoA. Lastly, aspartate derived from protein degradation forms oxaloacetate by transamination reaction, or fumarate via the reactions of the purine nucleotide cycle and of the urea cycle.

There is good evidence that the total concentration of CAC intermediates can vary by up to a few fold during transitions between metabolic situations. This is observed (i) in muscle during the transition from rest to exercise, (ii) in heart and liver upon supply of anaplerotic substrates, and (iii) in liver during the transition from fasting to feeding. However, in a given metabolic situation, anaplerotic flux in excess of physiological leakage must be balanced by a corresponding cataplerotic flux. For example, in the liver, gluconeogenic carbons of pyruvate and propionyl-CoA, which enter the CAC via pyruvate carboxylase and methylmalonyl-CoA mutase, leave the cycle via phosphoenolpyruvate (PEP) carboxykinase. In the perfused rat heart, anaplerosis from high concentrations of propionate is balanced by an efflux of malate. As anaplerotic molecules pass through reactions of the CAC, there is no net CO2 production, except from C1 of glutamine/glutamate. Thus, except for the latter, the production of labelled CO2 from a labelled anaplerotic substrate reflects not net oxidation of the substrate but isotopic exchanges in the CAC. This is true unless additional reactions form labelled mitochondrial acetyl-CoA from the anaplerotic substrate, for example via malic enzyme+ pyruvate dehydrogenase, or PEP carboxykinase and pyruvate dehydrogenase.

In addition to maintaining the pool of CAC intermediates, anaplerotic and cataplerotic reactions play a central role in gluconeogenesis from amino acids, lactate and pyruvate, as well as in the urea cycle. In the brain, anaplerotic reactions contribute to the regulation of the metabolism of neurotransmitters. Also, anaplerotic glutamine synthesis is coupled to removal of nitrogen from the brain in hyperammonaemia. Lastly, anaplerosis appears to play an important role in the secretion of insulin by pancreatic β-cells. The stimulation of anaplerosis by physiological fuel secretagogues is accompanied by a corresponding cataplerosis that transfers CAC intermediates to the extramitochondrial space. The transferred molecules could act as secretagogues or as exporters of equivalents of NADPH, acetyl-CoA or malonyl-CoA.

There is good evidence that a number of pathological conditions could benefit from anaplerotic therapy. Conditions associated with reperfusion injury (myocardial infarction, stroke, organ transplantation) are associated with damage to cell membranes and possible decrease in the pools of some CAC intermediates. Applications of anaplerotic therapy to the dietary treatment of some inherited metabolic diseases are extensively discussed in a paper published in this issue. Note that, when considering anaplerotic therapy, it is impossible in most cases to demonstrate either a decrease in the concentration of CAC intermediates in tissues or an excessive leakage of intermediates from a tissue. The latter might be inferred from evidence of the leakage of large molecules such as creatine kinase during myocardial infarction or rhabdomyolysis. Still, one cannot assess whether the concentration of oxaloacetate, the acetyl-CoA acceptor and least abundant CAC intermediate, is sufficient to allow proper CAC flux and energy production. In most cases, evidence of the need for anaplerotic therapy can only be inferred a posteriori from the improvement of cardiac function, muscle strength, or neurological status.



Henri Brunengraber, Charles R. Roe. J Inherit Metab Dis (2006) 29:327–331