The Key Role of Anaplerosis and Cataplerosis for Citric Acid Cycle Function

tricarboxylic acid cycle phosphoenolpyruvate carboxykinase phosphoenolpyruvate The oxidation of acetyl-CoA to CO2by the TCA1 cycle is the central process in energy metabolism. However, the TCA cycle also functions in biosynthetic pathways in which intermediates leave the cycle to be converted primarily to glucose, fatty acids, or non-essential amino acids. If TCA cycle anions are removed from the cycle they must be replaced to permit its continued function. This process is termed anaplerosis. Pyruvate carboxylase, which generates oxalacetate directly in the mitochondria, is the major anaplerotic enzyme. Conversely, 4- and 5-carbon intermediates enter the TCA cycle during the catabolism of amino acids. Because the TCA cycle cannot fully oxidize 4- and 5-carbon compounds, these intermediates must be removed from the cycle by a process termedcataplerosis. Cataplerosis may be linked to biosynthetic processes such as gluconeogenesis in the liver and kidney cortex, fatty acid synthesis in the liver, and glyceroneogenesis in adipose tissue. Cataplerotic enzymes present in many mammalian tissues include P-enolpyruvate carboxykinase (PEPCK), glutamate dehydrogenase, aspartate aminotransferase, and citrate lyase. In this review we have evaluated the roles of anaplerosis and cataplerosis in whole body metabolism. The expression anaplerotic sequences was a term used in biochemistry by Sir Hans Kornberg (1Kornberg H.L. Essays Biochem. 1966; 2: 1-31Crossref Scopus (359) Google Scholar) to describe a series of enzymatic reactions or pathways that replenish the pools of metabolic intermediates in the TCA cycle. These intermediates are critical for the functioning of the TCA cycle, the primary role of which is the oxidation of acetyl-CoA to carbon dioxide. The pool of TCA cycle intermediates is sufficient to sustain the oxidative carbon flux over a fairly wide range, so that during high energy consumption (e.g. exercise) or during lower energy consumption (e.g. fasting), there is not a large change in the pool size of TCA intermediates (2Graham T.E. Gibala M.J. Richter E.A. Galbo H. Kiens B. Saltin B. Skeletal Muscle Metabolism in Exercise and Diabetes. Plenum Press, New York1998: 271-286Google Scholar). However, in several physiological states, there is a large influx (anaplerosis) of 4- and 5-carbon intermediates into the TCA cycle. Because the TCA cycle cannot act as a carbon sink, anaplerosis must be coupled with the exit of intermediates from the cycle via cataplerosis. The importance of anaplerotic reactions for cellular metabolism is thus apparent. However, the coupling of this process with cataplerosis and the roles that both pathways play in the regulation of amino acid, glucose, and fatty acid metabolism have not been emphasized to a sufficient extent. The terms anaplerosis and cataplerosis describe reciprocal and correlative reactions involved in the function of the TCA cycle. The enzymatic steps in these processes have long been known, but the overall concept of a linkage between anaplerosis and cataplerosis should be underscored, because the balance between these two processes controls the entry and exit of TCA cycle anions. Anaplerotic and cataplerotic reactions are involved in the ultimate disposal of all metabolic intermediates. The metabolic role of anaplerosis and cataplerosis in amino acid metabolism varies with specific organs and is dependent on the nutritional/metabolic status of the individual. During feeding, the intestine is an important site of catabolism of enterally derived amino acids, whereas in the starved state amino acid catabolism occurs primarily in the kidney, liver, and muscle. The catabolism of amino acids produces gluconeogenic or ketogenic precursors (Table I). The disposal of gluconeogenic anions in the TCA cycle employs anaplerotic and cataplerotic pathways for their terminal oxidation. The only known pathway for the terminal oxidation of leucine is through acetoacetate to acetyl-CoA and subsequent oxidation in the TCA cycle. However, other amino acids also have for their disposal alternate ketogenic pathways for terminal oxidation. Thus, the ketogenic amino acids from proteolysis can be terminally oxidized in muscle, whereas the gluconeogenic amino acids are dependent upon anaplerosis and cataplerosis for conversion to glucose in the liver and kidney before oxidation to CO2 and H2O.Table IMetabolic fates of amino acids in the TCA cycle1. Amino acids converted to pyruvate Alanine, serine, glycine, threonine, cysteine, tryptophan2. Amino acids converted to oxaloacetate Aspartate, asparagine3. Amino acids converted to α-ketoglutarate Glutamate, glutamine, proline, histidine, arginine4. Amino acids converted to fumarate Phenylalanine, tyrosine5. Amino acids converted to succinyl-CoA Methionine, isoleucine, valine6. Amino acids converted to acetyl-CoA Leucine, isoleucine, lysine, phenylalanine, tyrosine, tryptophan, threonine Open table in a new tab The first reaction of the TCA cycle, citrate synthase, catalyzes the condensation of oxalacetate with acetyl-CoA; the oxalacetate is subsequently regenerated by the reactions of the cycle and condenses with another molecule of acetyl-CoA. However, the TCA cycle also functions in biosynthetic processes in which intermediates are removed from the cycle; this necessitates anaplerotic reactions to replenish TCA cycle intermediates to ensure its continued function. Pyruvate carboxylase, which synthesizes oxalacetate from pyruvate in the mitochondrial matrix, is the archetypical anaplerotic enzyme. The activity of this enzyme is high in many tissues (e.g. 10–12 units/g of liver); acetyl-CoA is a positive allosteric regulator of the enzyme. Anaplerosis is obligatory during both gluconeogenesis and lipogenesis when malate (gluconeogenesis) or citrate (lipogenesis) leaves the mitochondria and is further metabolized to form glucose or fatty acids, respectively. If intermediates can be added to the TCA cycle, it is equally important to remove them to avoid the accumulation of anions in the mitochondrial matrix. Cataplerosis describes reactions involved in the disposal of TCA cycle intermediates. There are several cataplerotic enzymes; these include PEPCK, aspartate aminotransferase, and glutamate dehydrogenase. Each of these reactions has as substrate a TCA cycle anion that is converted to a product that effectively removes intermediates from the cycle. In the liver and kidney, the role of PEPCK in cataplerosis is of special importance because it is a common route for the generation of PEP from oxalacetate to be used for gluconeogenesis. Alternatively, in muscle, PEP can be converted to pyruvate that can be decarboxylated to acetyl-CoA for subsequent oxidation to CO2 in the TCA cycle. The regulation of anaplerosis and cataplerosis depends upon the metabolic and physiologic state and the specific tissue/organ involved. For example, during starvation, cataplerosis via phosphoenolpyruvate to support gluconeogenesis may be regulatory in the liver, whereas in the kidney anaplerosis via uptake of glutamine may be regulatory. Anaplerotic and cataplerotic intermediates entering and exiting the TCA cycle are shown in Fig. 1. A detailed and elegant analysis of amino acid metabolism can be found in a review by Jungas et al. (3Jungas R.L. Halperin M.L. Brosnan J.T. Physiol. Rev. 1992; 72: 419-448Crossref PubMed Scopus (234) Google Scholar). The interplay between anaplerotic and cataplerotic reactions in humans was demonstrated by renal metabolism during total, prolonged starvation (4Owen O.E. Felig P. Morgan A.P. Wahren J. Cahill Jr., G.F. J. Clin. Invest. 1969; 48: 584-594Crossref PubMed Scopus (569) Google Scholar). Arteriovenous concentration differences of metabolites across the kidneys coupled with urinary nitrogen losses showed that the kidney extracted glutamine and produced urinary ammonium (5Van Slyke D.D. Phillips R.A. Hamilton P.B. Archibald R.M. Futcher P.H. Hiller A. J. Biol. Chem. 1943; 150: 481-482Abstract Full Text PDF Google Scholar). Concurrently, the kidney released glucose into the blood. It was initially recognized that renal ammoniagenesis was related to ketonuria during prolonged starvation when there is an increase in ketogenesis (6Sapir D.G. Owen O.E. Cheng J.T. Ginsberg R. Boden G. Walker W.G. J. Clin. Invest. 1972; 51: 2093-2102Crossref PubMed Scopus (26) Google Scholar). However, it was not generally appreciated that the entry (anaplerosis) and removal (cataplerosis) of intermediates into and out of the TCA cycle as related to renal ammoniagenesis and gluconeogenesis had to be balanced. This fundamental principle is poorly understood and is the foundation of this paper. During prolonged starvation glutamine is transported from muscle to the kidney where the amino and amide groups are used for ammonia formation. The ammonia released from the renal cells serves to titrate the acidity of the tubular urine created by the disassociation of organic acids, primarily β-hydroxybutyric and acetoacetic acids. For ammonia generation to continue, glutamine undergoes anaplerotic reactions to form α-ketoglutarate that enters the TCA cycle and is sequentially converted to malate that leaves the mitochondria. Malate is oxidized in the cytosol to oxalacetate that is subsequently converted to PEP and then to glucose. Thus, anaplerotic and cataplerotic reactions are essential and balanced during renal ammoniagenesis and gluconeogenesis. The heightened ketonuria that occurs with ketonemia is related to the need for the kidney to generate glucose during total starvation when renal gluconeogenesis accounts for about 50% of the net glucose synthesis (4Owen O.E. Felig P. Morgan A.P. Wahren J. Cahill Jr., G.F. J. Clin. Invest. 1969; 48: 584-594Crossref PubMed Scopus (569) Google Scholar, 7Owen O.E. Smalley K.J. D'Alessio D.A. Mozzoli M.A. Dawson E.K. Am. J. Clin. Nutr. 1998; 68: 12-34Crossref PubMed Scopus (90) Google Scholar). Thus, renal ammoniagenesis and gluconeogenesis are tightly interlocked and dependent upon balanced anaplerotic reactions to replenish the α-ketoglutarate in the TCA cycle and cataplerotic reactions to drain remnant 4-carbon metabolic intermediates from the cycle to synthesize glucose (7Owen O.E. Smalley K.J. D'Alessio D.A. Mozzoli M.A. Dawson E.K. Am. J. Clin. Nutr. 1998; 68: 12-34Crossref PubMed Scopus (90) Google Scholar). In addition, there is a metabolic bonus when the kidneys excrete urinary ammonium during starvation. The caloric value of protein is greater when amino acid nitrogen is lost in the urine as ammonium rather than urea because it requires four molecules of ATP to generate a molecule of urea via the urea cycle. In addition, energy is required for the synthesis of creatine and uric acid. Despite the relatively slow rate of turnover of skeletal muscle protein, it represents the largest reservoir of amino acids because of its large mass. Following an overnight fast, there is a net release of amino acids from skeletal muscle; however, the amino acids released do not reflect the amino acid composition of the skeletal muscle proteins (3Jungas R.L. Halperin M.L. Brosnan J.T. Physiol. Rev. 1992; 72: 419-448Crossref PubMed Scopus (234) Google Scholar, 8Felig P. Bergman M. Felig P. Baxter J.D. Frohman L.A. Endocrinology and Metabolism. 3rd Ed. McGraw-Hill, Inc., New York1995: 1107-1250Google Scholar). This suggests that there is local metabolism and interconversion of amino acids in the muscle. Specifically, alanine and glutamine represent a disproportionately larger fraction of amino acids released by the skeletal muscle when compared with the amino acid composition of skeletal muscle proteins. The relative proportion of these amino acids released by muscle also changes with the metabolic status, such as prolonged starvation or diabetes, or in response to administration of insulin or glucagon (3Jungas R.L. Halperin M.L. Brosnan J.T. Physiol. Rev. 1992; 72: 419-448Crossref PubMed Scopus (234) Google Scholar, 8Felig P. Bergman M. Felig P. Baxter J.D. Frohman L.A. Endocrinology and Metabolism. 3rd Ed. McGraw-Hill, Inc., New York1995: 1107-1250Google Scholar, 9Reeds P.J. Fjeld C.R. Jahoor F. J. Nutr. 1994; 124: 906-910Crossref PubMed Scopus (282) Google Scholar, 10Castillo C.E. Katz A. Spencer M.K. Yan Z. Nyomba B.L. Am. J. Physiol. 1991; 261: E598-E605PubMed Google Scholar, 11Gibala M.J. MacLean D.A. Graham T.E. Saltin B. Am. J. Physiol. 1998; 275: E235-E242PubMed Google Scholar). These data suggest that a local metabolism of amino acids occurs in the skeletal muscle that results in the de novo synthesis of certain non-essential amino acids, primarily alanine and glutamine. Arteriovenous concentration differences across skeleton muscles show net uptake and/or release of lactate, ammonia, alanine, glutamine, and glutamate at rest and during exercise (11Gibala M.J. MacLean D.A. Graham T.E. Saltin B. Am. J. Physiol. 1998; 275: E235-E242PubMed Google Scholar). In addition, during exercise there is an increase in the TCA cycle intermediates; however, the increase in concentration is not equal in all the TCA intermediates (Table II).Table IIIntramuscular concentrations of individual TCA cycle intermediates at rest and during exerciseRest5 min10 minCitrate0.362 ± 0.0470.658 ± 0.0772-ap ≤ 0.05versus rest.0.631 ± 0.0522-ap ≤ 0.05versus rest.Isocitrate0.085 ± 0.0130.194 ± 0.0222-ap ≤ 0.05versus rest.0.200 ± 0.0222-ap ≤ 0.05versus rest.α-Ketoglutarate0.050 ± 0.0040.036 ± 0.0052-ap ≤ 0.05versus rest.0.038 ± 0.0052-ap ≤ 0.05versus rest.Succinate0.368 ± 0.0760.567 ± 0.1150.609 ± 0.118Fumarate0.087 ± 0.0060.198 ± 0.0292-ap ≤ 0.05versus rest.0.195 ± 0.0322-ap ≤ 0.05versus rest.Malate0.365 ± 0.0371.163 ± 0.2032-ap ≤ 0.05versus rest.1.182 ± 0.1602-ap ≤ 0.05versus rest.Oxalacetate0.012 ± 0.0030.030 ± 0.0052-ap ≤ 0.05versus rest.0.027 ± 0.0062-ap ≤ 0.05versus rest.2-a p ≤ 0.05versus rest. Open table in a new tab During caloric restriction, amino acids also provide a source of energy. Amino acids yield part of their energy during oxidative deamination, but their carbon skeleton must undergo subsequent catabolism to be fully metabolized. Although most amino acids enter the TCA cycle as 4- or 5-carbon compounds, only acetyl-CoA produced from their catabolism can be fully oxidized in the cycle. Recent data from studies in humans have shown that only a small increase in the concentration of TCA intermediates (mostly 4-carbon) occurs during fasting or exercise, thus confirming the concept that only small changes in the amounts of these intermediates are required to adapt to the need for energy (12Sahlin K. Katz A. Broberg S. Am. J. Physiol. 1990; 259: C834-C841Crossref PubMed Google Scholar, 13Garber A.J. Karl I.E. Kipnis D.M. J. Biol. Chem. 1976; 251: 826-835Abstract Full Text PDF PubMed Google Scholar). The removal (cataplerosis) of TCA cycle anions generated from the entry of amino acids occurs via the action of PEPCK, glutamate dehydrogenase, or aspartate aminotransferase. Alanine and glutamine are synthesized from other amino acids and released into the circulation. Alanine is generated by the transamination of pyruvate via alanine aminotransferase. The possible sources of pyruvate are glucose and lactate, or PEP via PEPCK, using as a substrate the oxalacetate generated in the TCA cycle (cataplerosis) (13Garber A.J. Karl I.E. Kipnis D.M. J. Biol. Chem. 1976; 251: 826-835Abstract Full Text PDF PubMed Google Scholar). The carbon skeleton for the synthesis of glutamine can be generated from the TCA cycle intermediates formed by the catabolism of gluconeogenic amino acids such as aspartate and asparagine (14Chang T.W. Goldberg A.L. J. Biol. Chem. 1978; 253: 3685-3695Abstract Full Text PDF PubMed Google Scholar). These amino acids are capable of generating intermediates that can be converted by forward flow of the TCA cycle to the α-ketoglutarate required for glutamate synthesis. Transamination of α-ketoglutarate, using the branched-chain amino acids as the source of the amino groups, accounts for the synthesis of glutamate, which is then converted to glutamine by glutamine synthase using ammonia generated in muscle by the purine nucleotide cycle. In light of the concept of balanced anaplerosis and cataplerosis, branched-chain amino acid metabolism in muscle needs to be studied further. Another paradigm for the metabolic roles of anaplerosis and cataplerosis is the oxidation of glutamine to CO2 in the small intestine. In this case, the α-ketoglutarate formed from glutamine is converted to malate by the TCA cycle; the malate leaves the mitochondria and is oxidized in the cytosol to oxalacetate by NAD:malate dehydrogenase. The oxalacetate is decarboxylated to PEP by PEPCK, and the PEP is converted to pyruvate by pyruvate kinase. The pyruvate re-enters the mitochondria where it is decarboxylated to acetyl-CoA by the pyruvate dehydrogenase complex; the acetyl-CoA is then oxidized by the TCA cycle. Alternately, a fraction of the pyruvate may be transaminated to alanine in the cytosol (Fig.2). Recent research on the metabolism of glutamine in human adults and infants illustrates the critical role of anaplerosis and cataplerosis in amino acid metabolism in the gut. In normal healthy adults, almost 74% of the enterally administered glutamine was extracted by the splanchnic compartment during the first pass (15Matthews D.E. Marano M.A. Campbell R.G. Am. J. Physiol. 1993; 264: E848-E854PubMed Google Scholar), whereas 70–80% of enterally administered 13Cglutamine tracer was found in respiratory CO2 (16Haisch M. Fukagawa N.K. Matthews D.E. Am. J. Physiol. 2000; 278: E593-E602Crossref PubMed Google Scholar). Other data from studies of newborn infants also show that enterally administered glutamine is rapidly metabolized in the gut as an energy source for the enterocytes, resulting in increased urea production (17Devapatla S. Parimi P. O'Brien A. Gruca L. Kalhan S. Pediatr. Res. 2002; 51: 286AGoogle Scholar). The majority of the glutamine is metabolized by the small intestine and not the liver, because there is no observed dilution of parenterally administered 15Nglutamine. This suggests that the carbon skeleton of dietary glutamine is oxidized by the small intestine (not the liver) as a source of energy. The metabolism of glutamine by the small intestine illustrates the importance of both anaplerosis and cataplerosis in the metabolism of the 5-carbon intermediates formed from the catabolism of the carbon skeletons of amino acids. For the carbon skeleton of glutamine to be metabolized to CO2 in the TCA cycle, the α-ketoglutarate formed from glutamate (originally from the deamination of glutamine) must be converted to acetyl-CoA for full oxidation. As shown in Fig. 2, the removal of carbon from the TCA cycle involves the cataplerotic activity of PEPCK in the small intestine, which synthesizes PEP from the oxalacetate generated from the oxidation of α-ketoglutarate. The synthesis of triglyceride in adipose tissue during starvation is another example of cataplerosis linked to a biosynthetic pathway. There is a net breakdown of triglyceride in adipose tissue during fasting (lipolysis) that is stimulated by cAMP and inhibited by insulin. During starvation when the rates of lipolysis are highest, a major fraction (up to 30%) of the free fatty acids generated from triglyceride breakdown is re-esterified back to triglyceride in adipose tissue (18Elia M. Zed C. Neale G. Livesey G. 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Chem. 1967; 242: 2746-2750Abstract Full Text PDF PubMed Google Scholar, 23Reshef L. Hanson R.W. Ballard F.J. J. Biol. Chem. 1970; 245: 5979-5984Abstract Full Text PDF PubMed Google Scholar). Rats fed a high protein, carbohydrate-free diet synthesize up to 80% of their glyceride glycerol in white adipose tissue by glyceroneogenesis (24Botion L.M. Brito M.N. Brito N.A. Brito S.R. Kettelhut Metabolism. 1998; Full Text PDF PubMed Scopus Google Scholar). is also an important pathway for the synthesis of triglyceride during It has been that of the fatty acids up by the liver during fasting is converted to triglyceride and released as (7Owen O.E. Smalley K.J. D'Alessio D.A. Mozzoli M.A. Dawson E.K. Am. J. Clin. Nutr. 1998; 68: 12-34Crossref PubMed Scopus (90) Google Scholar). is a major pathway for the synthesis of the 3-glycerol phosphate that is required to support triglyceride synthesis in the by et al. (24Botion L.M. Brito M.N. Brito N.A. Brito S.R. Kettelhut Metabolism. 1998; Full Text PDF PubMed Scopus Google Scholar) have that glyceroneogenesis in the of fed a high protein, carbohydrate-free diet 80% of the glyceride glycerol in In humans have for of the glyceride glycerol is derived from glyceroneogenesis and only from glycerol S. L. Hanson R.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The TCA cycle is balanced between the and of intermediates for metabolic The of the TCA cycle as a needs in light of its role in The cycle as a on a in which the flow of into the must be balanced by the flow out or the be with In this we have several metabolic in which the two anaplerosis and cataplerosis, to ensure the balance of carbon flow into and out of the TCA cycle. The of this fundamental is in its and in its It is as in