Glutamate, at the Interface between Amino Acid and Carbohydrate Metabolism

The liver is the major site of gluconeogenesis, the major organof amino acid catabolism and the only organ with a completeurea cycle. These metabolic capabilities are related, and theserelationships are best exemplified by an examination of thedisposal of the daily protein load. Adults, ingesting a typicalWestern diet, will consume ~100 g protein/d; the great bulkof this is metabolized by the liver. Although textbooks suggestthat these amino acids are oxidized in the liver, total oxidationcannot occur within the confines of hepatic oxygen uptake andATP homeostasis. Rather, most amino acids are oxidized only partiallyin the liver, with the bulk of their carbon skeleton being convertedto glucose. The nitrogen is converted to urea and, to a lesserextent, to glutamine. The integration of the urea cycle with gluconeogenesisensures that the bulk of the reducing power (NADH) requiredin the cytosol for gluconeogenesis can be provided by ancillaryreactions of the urea cycle. Glutamate is at the center of thesemetabolic events for three reasons. First, through the well-describedtransdeamination system involving aminotransferases and glutamatedehydrogenase, glutamate plays a key catalytic role in the removalof {alpha}

-amino nitrogen from amino acids. Second, the "glutamatefamily" of amino acids (arginine, ornithine, proline, histidineand glutamine) require the conversion of these amino acids to glutamatefor their metabolic disposal. Third, glutamate serves as substratefor the synthesis of N-acetylglutamate, an essential allostericactivator of carbamyl phosphate synthetase I, a key regulatoryenzyme in the urea cycle.

KEY WORDS: • gluconeogenesis • urea synthesis • liver metabolism • dietary protein • glutamate • glutamine

INTRODUCTION

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INTRODUCTION

In considering relationships between glutamate and carbohydrate metabolism,this paper will focus exclusively on gluconeogenesis. However,for physiologic relevance, it is important not to consider glutamatealone, but glutamate in the context of the metabolism of all ofthe amino acids. Second, it is important not to consider gluconeogenesisalone, but other pathways with which it is integrated, in particular,the urea cycle. Therefore, this paper examines the metabolicdisposal of the dietary protein load and considers the specificrole of glutamate in this process under the following three headings:1) the key role of glutamate and of glutamate dehydrogenasein transdeamination of amino acids; 2) the metabolism of theglutamate family of amino acids; and 3) the synthesis of N-acetylglutamate.The remarkable metabolic versatility of glutamate will alsobe discussed.

Metabolic disposal of dietary protein

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Metabolic disposal of dietary...

An active, healthy adult male, eating a typical Western diet,will ingest ~100 g of protein per day. Assuming that the aminoacid composition of this protein is similar to that in meatsand that digestion is highly efficient, the body will be facedwith disposing ~1000 mmol of amino acids per day. Such an adultwill be in nitrogen balance so that the rates of protein synthesisand of protein degradation will be equal, over 24 h. Thus the1000 mmol of amino acids must be oxidized. The liver is themajor organ of amino acid metabolism; it is frequently statedthat the liver is responsible for the oxidation of dietary aminoacids. This statement is incorrect for two reasons. First, itignores the fact that extrahepatic tissues (in particular, theintestine, muscle and kidney) are quantitatively important inthe disposal of some specific amino acids. Often, however, aminoacid metabolism in extrahepatic tissues produces other amino acids(e.g., renal glycine metabolism produces serine; intestinal glutaminemetabolism produces alanine) that must be metabolized by the liver.Thus, the liver is faced with metabolizing ~900 mmol of amino acidsper day. The second reason why it is incorrect to state thatthe liver is responsible for the oxidation of amino acids relatesto the term "oxidation." The liver does, indeed, metabolize~900 mmol of amino acids per day, but it does not oxidize themcompletely. Indeed, it cannot oxidize them within the confinesof its oxygen consumption and energy requirements. These mattershave been considered extensively by Jungas et al. (1992)

An obvious, but key fact about the oxidation of amino acidsis that it will consume oxygen and produce ATP. The oxidationof the 900 mmol of amino acids by the liver would consume ~3.8mol O₂/d and would produce ~22 mol ATP (Jungas et al. 1992 ).We now appreciate that the actual ATP yield will be somewhatlower than this theoretical value because of normal proton leakageacross the mitochondrial membrane, either through the lipidbilayer or through uncoupling proteins (Rolfe and Brown 1997 ).However, the O₂ consumption required for the complete oxidationof these 900 mmol of amino acids cannot change, because it isdetermined by the stoichiometry of the oxidation reactions.The liver consumes ~50 mL O₂/min or ~3 mol O₂/d (Hagenfeldtet al. 1980 ). Thus, even if no other substrate were consumedby the liver (i.e., no dietary fat or carbohydrate or alcoholwere oxidized), it would not be possible for the liver to oxidize900 mmol of amino acids per day to CO₂ and water. Because itis certain that many other substrates are oxidized by the liver,the only viable conclusion is that liver amino acid metabolism,even in the prandial state, involves their conversion to glucoseand ketones (Jungas et al. 1992 ). Even the conversion of theseamino acids to glucose and urea is an oxygen-consuming process(~1.4 mol of O₂/d). Gluconeogenesis is often referred to asan ATP-consuming process; this is true of substrates such aslactate when all three carbons of the precursor molecule areconverted to glucose. But gluconeogenesis from a physiologicmixture of amino acids involves the necessary oxidation of someof the carbons to CO₂ (with the consumption of oxygen and productionof ATP) as well as the partial reduction of some of the carbonsto glucose and the synthesis of urea. The net ATP balance is closeto zero because the ATP produced by oxidative reactions is balancedby the ATP used in urea synthesis and in the gluconeogenic pathway.There is also close redox balance between the two processesso that the bulk of the NADH required in the cytosol for gluconeogenesis isprovided by reactions ancillary to the urea cycle. Of course, glycogen,rather than glucose, may be one of the immediate products of thisgluconeogenesis. It is now clear that fasting gluconeogenesisin humans continues for some time after a meal, with glycogenas the product (Shulman and Landau 1992 ). It is also known that,in experimental animals, synthesis of glycogen via the "indirectpathway" after a fast is increased if the meal is rich in protein(Rosetti et al. 1989 ).

Key roles of glutamate in the metabolism of dietary protein

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Key roles of glutamate...

Glutamate is at the center of the disposal of the daily protein loadfor three reasons. First, there is the "glutamate family" of aminoacids. These amino acids (glutamate, glutamine, proline, histidine,arginine and ornithine) comprise ~25% of the dietary amino acidintake and will be disposed of via conversion to glutamate. Second,there is the key role of glutamate dehydrogenase together with theglutamate-linked aminotransferases in effecting the removalof {alpha}

-amino nitrogen from almost all of the amino acids via transdeamination.Third, there is the key role of glutamate in N-acetylglutamatesynthesis, which ensures that the rate of urea synthesis isin accord with rates of amino acid deamination.

The "glutamate family" of amino acids

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The "glutamate family" of...

These amino acids (glutamate, glutamine, proline, histidine, arginineand ornithine) have always been considered as a group because theirmetabolism converges on that of glutamate itself. However, itis now quite apparent that rather significant differences existbetween them with respect to the tissue and cellular locationof their metabolic disposal. Dietary glutamate is metabolizedto a great extent in the gastrointestinal tract. The more recentdata are particularly persuasive in that they show quite clearly,in fed infant pigs, that almost no enteral [¹³C₅] glutamateappeared in portal venous blood (Reeds et al. 1996

). Possibleproducts of gastrointestinal metabolism are alanine, arginine,proline and glutathione, but the quantitation of these productsremains to be clarified. The situation with glutamine is morecomplex. Stumvoll et al. (1998)

argued that the kidney, ratherthan the liver, is the primary organ of glutamine metabolism(especially, gluconeogenesis from glutamine) in fasting humans.We have shown that the kidneys of rats fed a high protein diet extractsubstantial quantities of glutamine and have increased activitiesof renal glutaminase (Brosnan et al. 1978

, Brosnan 1987

).It is evident that this renal glutamine metabolism is relatedto the production of urinary ammonia, which is used to facilitatethe excretion of metabolic acids (principally, sulfuric acid)that arise from the catabolism of the sulfur-containing aminoacids, methionine and cysteine. Thus, administration of sodiumbicarbonate, sufficient to neutralize these acids greatly reducesthe renal uptake and metabolism of glutamine. Of course, theintestine is also a major consumer of glutamine (Windmuellerand Spaeth 1980

). Proline and histidine catabolism have notbeen studied extensively, but it is clear that they occur, primarilyin the liver (Jungas et al. 1992

Considerable new information has appeared recently on the locationof arginine catabolism. Some arginine catabolism occurs in theintestine (Windmueller and Spaeth 1976 ), but liver is the principalsite. We now know that hepatic arginine catabolism is confinedto the perivenous hepatocytes. This conclusion is founded on twopieces of evidence, i.e., the demonstration by Kuo et al. (1991) that hepatic ornithine aminotransferase is restricted to thesame small population of hepatocytes proximal to the terminal hepaticvein in which glutamine synthetase is found, and our own demonstrationthat arginine catabolism occurs in the isolated, nonrecirculating,retrogradely perfused liver (O’Sullivan et al. 1998 ).By this means, the liver maintains spatial separation of thetwo major pathways of hepatic arginine metabolism because theurea cycle is restricted to the periportal hepatocytes.

The role of glutamate in transamination and deamination

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The role of glutamate...

The crucial role of glutamate/ {alpha}

-ketoglutarate as transamination partnersis well known. We now know that all of the common amino acids, exceptfor lysine, may be transaminated. Similarly, the key role of glutamatedehydrogenase, in conjunction with the transaminases, in the transdeaminationof amino acids is well established. These enzymes are freelyreversible so that they also afford a mechanism for the synthesisof the nonessential amino acids.

N-Acetylglutamate synthesis

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N-Acetylglutamate synthesis

The rate of urea synthesis must be regulated sensitively with respectto the rate of amino acid deamination. Simple regulation by substrateconcentration is too crude a mechanism for this key process. Forexample, a threefold increase in urea synthesis would require(at least) a threefold increase in ammonia concentration, assuming Michaelis-Mentenkinetics. Homeostasis would be served very poorly by a situationin which one would require a threefold increase in the concentrationof toxic ammonia to effect a threefold increase in its disposal.The regulation of the urea cycle is brought about, chronically,by an adjustment in the amount of urea cycle enzymes as dietaryprotein varies (Schimke and Doyle 1970

) and, acutely, at thelevel of carbamoylphosphate synthetase-I. As an obligatory activator,this enzyme requires N-acetylglutamate, which is synthesizedwithin liver mitochondria from glutamate and acetyl-CoA. Theconcentration of N-acetylglutamate can change quite rapidlyto facilitate increased flux through the urea cycle (Meijeret al. 1990

). One way in which N-acetylglutamate levels are regulatedis through arginine, which is known to activate N-acetylglutamatesynthetase. We must also consider that N-acetylglutamate synthesismay be regulated via increased provision of glutamate as a resultof activation of glutaminase. Hepatic glutaminase has the remarkableproperty of being activated by its own product, ammonia (Curthoysand Watford 1995

). Feedback activation is bizarre, unstableand unsustainable. Perhaps the best everyday example of feedbackactivation is an explosion in which the detonation of a smallquantity of an explosive provides sufficient heat to detonatethe rest of the material. What could possibly be the functionof such a metabolic control system? The properties of glutaminase,in isolation, do not provide an answer to this conundrum; ananswer may possibly arise if one considers that an important functionof hepatic glutaminase is to provide intramitochondrial glutamatefor N-acetylglutamate synthesis. Thus, when the hepatic ammoniaconcentration tends to increase, it would activate glutaminasewhich, by providing glutamate to N-acetylglutamate synthetase,increases intramitochondrial concentrations of N-acetylglutamate;these in turn, by activating carbamoylphosphate synthetase I,will actually effect the removal of ammonia.

The metabolic versatility of glutamate

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The metabolic versatility of...

In almost all cells. the intracellular concentration of glutamate ismaintained at quite high concentrations compared with its concentrationin extracellular fluids. Typically, intracellular concentrationsof 2–5 mmol/L are common, compared with extracellular concentrationsof ~0.05 mmol/L. Our own data show that glutamate is one ofthe most abundant amino acids in liver, kidney, skeletal muscle andbrain (Brosnan et al. 1983

). Such high concentrations pointto the important roles glutamate plays in all tissues. In additionto its role as a key transamination partner, glutamate is alsorequired for the synthesis of glutathione, a key component inour defenses against oxidative stresses. Glutamate is also involvedin the glutamate/aspartate shuttle, which effects the oxidationof cytoplasmically produced NADH in many cells. Finally, glutamate,by virtue of being readily convertible to {alpha}

-ketoglutarate bymeans of a variety of reversible transaminases, can serve an anapleroticfunction for the Krebs’ cycle. No other amino acid displayssuch remarkable metabolic versatility.

FOOTNOTES

^¹ Presented at the International Symposium on Glutamate, October12–14, 1998 at the Clinical Center for Rare Diseases Aldoe Cele Daccó, Mario Negri Institute for PharmacologicalResearch, Bergamo, Italy. The symposium was sponsored jointlyby the Baylor College of Medicine, the Center for Nutritionat the University of Pittsburgh School of Medicine, the MonellChemical Senses Center, the International Union of Food Science andTechnology, and the Center for Human Nutrition; financial support wasprovided by the International Glutamate Technical Committee.The proceedings of the symposium are published as a supplementto The Journal of Nutrition. Editors for the symposium publicationwere John D. Fernstrom, the University of Pittsburgh School ofMedicine, and Silvio Garattini, the Mario Negri Institute for PharmacologicalResearch.

^² Supported by a grant from the Medical Research Council of Canada.

REFERENCES

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