Margaret E. Brosnan

Branched-Chain Amino Acids: Enzyme and Substrate Regulation

The three branched-chain amino acids (BCAAs) are the most hydrophobic of the amino acids and play crucial roles in determining the structures of globular proteins as well as the interaction of the transmembrane domains of membranous proteins with phospholipid bilayers. However, the three BCAAs do not behave identically. In terms of protein secondary structure, valine and isoleucine exhibit a definite preference for the beta-structure, whereas leucine has a higher preference for the alpha-helix. Although mutation of one BCAA to another is commonly regarded as conservative, there are well-documented examples of such substitutions that have a significant effect on protein function. The occurrence of BCAA in nature is, therefore, attributable to their primary role in protein structure, not to their secondary metabolic roles. These functions are important for almost all proteins; therefore, BCAA commonly account for approximately 20-25% of most dietary proteins. Dietary BCAA largely escape first-pass splanchnic metabolism. The first steps in their catabolism are common to all three, involving the BCAA aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD). Their further metabolism employs distinct pathways to different end-products (glucose and/or ketone bodies). However, the fact that the flux-generating step for the catabolism of the three BCAAs occurs at one of the common steps indicates that the production of these downstream products are not individually regulated and, hence, may not play important individual roles. The catabolism of the BCAAs is highly regulated by both allosteric and covalent mechanisms. BCKD is inhibited by phosphorylation and activated by dephosphorylation. Allosteric inhibition of the kinase by the branched-chain keto acids (BCKA) (particularly by alpha-ketoisocaproate) serves both as a mechanism for promoting the catabolism of excess quantities of these amino acids as well as for conserving low concentrations of these dietary essential amino acids. Cytosolic and mitochondrial isoenzymes of BCAT have been identified. They are thought to play an important role in brain neurotransmitter metabolism.

Hepatic glutamate metabolism: a tale of 2 hepatocytes

Glutamate plays a central role in hepatic amino acid metabolism, both because of its role in the transdeamination of most amino acids and because the catabolism of arginine, ornithine, proline, histidine, and glutamine gives rise to glutamate. It is now appreciated that different hepatic functions are restricted to hepatocyte subpopulations within different acinar zones. This is also a feature of glutamate metabolism. Glutamine catabolism and synthesis are physically separated by zonation, with glutamine synthetase restricted to a narrow band of hepatocytes in zone 3 of the hepatic acinus, whereas glutaminase occurs in zone 1. Arginine and ornithine metabolism is also restricted to particular hepatocyte subpopulations. Ornithine aminotransferase, the regulated enzyme of arginine and ornithine catabolism, is restricted to the same zone 3 cells as glutamine synthetase, whereas the urea cycle is found in the remaining hepatocytes. This separation facilitates the independent regulation of these 2 different metabolic processes. We know the acinar localization of only a small fraction of the approximately 15,000 genes expressed in the liver. Knowledge of the acinar localization of metabolic processes is essential for an appreciation of their relation to other hepatic functions and their regulation.

Regulation of homocysteine metabolism.

We have used a combination of in vivo and in vitro techniques to measure factors regulating homocysteine metabolism and the plasma concentration of this atherogenic amino acid. The germane findings include: 1. Homocysteine metabolism in rat kidney proceeds predominantly through the transsulfuration pathway, whose enzymes are enriched within the proximal cells of kidney tubules. Furthermore, the rat kidney possesses significant reserve capacity to handle both acute and chronic elevations in plasma homocysteine concentrations. 2. Plasma homocysteine concentrations are lower in diabetic rats. Insulin administration corrects this perturbation. Therefore, insulin and/or one of its counter-regulatory hormones affects homocysteine metabolism, possibly through an increased flux in the hepatic transsulfuration pathway. In support of these data, glucagon administration to rats produced similar results. Further support was provided by studies with isolated rat hepatocytes, from which homocysteine export was reduced when incubated in the presence of glucagon.

Glutamate: a truly functional amino acid.

Glutamate is one of the most abundant of the amino acids. In addition to its role in protein structure, it plays critical roles in nutrition, metabolism and signaling. Post-translational carboxylation of glutamyl residues increases their affinity for calcium and plays a major role in hemostasis. Glutamate is of fundamental importance to amino acid metabolism, yet the great bulk of dietary glutamate is catabolyzed within the intestine. It is necessary for the synthesis of key molecules, such as glutathione and the polyglutamated folate cofactors. It plays a major role in signaling. Within the central nervous system, glutamate is the major excitatory neurotransmitter and its product, GABA, the major inhibitory neurotransmitter. Glutamate interaction with specific taste cells in the tongue is a major component of umami taste. The finding of glutamate receptors throughout the gastrointestinal tract has opened up a new vista in glutamate function. Glutamate is truly a functional amino acid.

Homocysteine metabolism in diabetes

An increase in the plasma level of Hcy (homocysteine), an intermediate in the catabolism of methionine, has been identified as a risk factor for many diseases including CVD (cardiovascular disease). CVD is the major cause of death in patients with diabetes mellitus. Therefore the study of Hcy metabolism in diabetes mellitus has been a major focus of current research. Studies conducted in our laboratory were able to show that in both Type 1 and Type 2 diabetes with no renal complications, the plasma Hcy levels were lower than in controls. In Type 1 diabetes, increased activities of the trans-sulfuration enzymes were the major cause for the reduction in plasma Hcy. In Type 2 diabetes, BHMT (betaine:homocysteine methyltransferase) was also observed to play a major role in the increased catabolism of Hcy in addition to the trans-sulfuration enzymes. We were also able to demonstrate the direct effect of insulin and the counter-regulatory hormones on the regulation of cystathionine beta-synthase and BHMT, which accounts for the changes in the activities of these two enzymes seen in diabetes mellitus.

Renal Arginine Metabolism

The kidney plays a major role in arginine metabolism in 3 principal ways: arginine synthesis, creatine synthesis, and arginine reabsorption. Appreciable quantities of arginine are synthesized in the kidney from citrulline produced by the intestine. The renal enzymes of arginine synthesis, argininosuccinate synthetase and argininosuccinate lyase, occur in the cells of the proximal tubule. The rate of arginine synthesis depends on citrulline delivery and does not appear to be regulated by dietary arginine availability. Renal arginine synthesis in humans produces approximately 2 g arginine/d, which may be compared to an intake, from a Western diet, of approximately 4 to 5 g/d. Spontaneous, nonenzymatic breakdown of creatine and creatine phosphate to creatinine causes the excretion of 1 to 2 g creatinine/d and requires the replacement of an equivalent amount of creatine from the diet and by endogenous synthesis. The first enzyme of creatine biosynthesis, L-arginine:glycine amidinotransferase, occurs in the kidney and produces guanidinoacetate, which is released into the renal vein. The renal output of guanidinoacetate, however, is rather low, and we propose that the entire pathway of creatine synthesis may also occur in the liver. Renal arginine reabsorption salvages approximately 3 g arginine/d. At the apical membrane of proximal tubular cells, arginine shares a transporter with lysine, ornithine, and cystine. Defects in this heteromeric transporter cause cystinuria, which is also characterized by urinary loss of arginine, lysine, and ornithine. Arginine is transported out of the proximal tubular cells at the basolateral membrane by another heteromeric transporter, which also transports lysine and ornithine. Defects in this transporter cause lysinuric protein intolerance.

Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo

Since creatinine excretion reflects a continuous loss of creatine and creatine phosphate, there is a need for creatine replacement, from the diet and/or by de novo synthesis. Creatine synthesis requires three amino acids, methionine, glycine, and arginine, and two enzymes, l-arginine:glycine amidinotransferase (AGAT), which produces guanidinoacetate acid (GAA), and guanidinoacetate methyltransferase (GAMT), which methylates GAA to produce creatine. In the rat, high activities of AGAT are found in the kidney, whereas high activities of GAMT occur in the liver. Rat hepatocytes readily convert GAA to creatine; this synthesis is stimulated by the addition of methionine, which increases cellular S-adenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. (15)N from (15)NH(4)Cl is readily incorporated into urea but not into creatine. Hepatic uptake of GAA is evident in vivo by livers of rats fed a creatine-free diet but not when rats were fed a creatine-supplemented diet. Rats fed the creatine-supplemented diet had greatly decreased renal AGAT activity and greatly decreased plasma [GAA] but no decrease in hepatic GAMT or in the capacity of hepatocytes to produce creatine from GAA. These studies indicate that hepatocytes are incapable of the entire synthesis of creatine but are capable of producing it from GAA. They also illustrate the interplay between the dietary provision of creatine and its de novo synthesis and point to the crucial role of renal AGAT expression in regulating creatine synthesis in the rat.

Creatine Synthesis Is a Major Metabolic Process in Neonatal Piglets and Has Important Implications for Amino Acid Metabolism and Methyl Balance

Our objectives in this study were as follows: 1) to determine the rate of creatine accretion by the neonatal piglet; 2) identify the sources of this creatine; 3) measure the activities of the enzymes of creatine synthesis; and 4) to estimate the burden that endogenous creatine synthesis places on the metabolism of the 3 amino acids required for this synthesis: glycine, arginine, and methionine. We found that piglets acquire 12.5 mmol of total creatine (creatine plus creatine phosphate) between 4 and 11 d of age. As much as one-quarter of creatine accretion in neonatal piglets may be provided by sow milk and three-quarters by de novo synthesis by piglets. This rate of creatine synthesis makes very large demands on arginine and methionine metabolism, although the magnitude of the demand depends on the rate of remethylation of homocysteine and of reamidination of ornithine. Of the 2 enzymes of creatine synthesis, we found high activity of l-arginine:glycine amidinotransferase in piglet kidneys and pancreas and of guanidinoacetate methyltransferase in piglet livers. Piglet livers also had appreciable activities of methionine adenosyltransferase, which synthesizes S-adenosylmethionine, and of betaine:homocysteine methyltransferase, methionine synthase, and methylene tetrahydrofolate reductase, which are required for the remethylation of homocysteine to methionine. Creatine synthesis is a quantitatively major metabolic process in piglets.

A Mass Isotopomer Study of Urea and Glutamine Synthesis from 15N-labeled Ammonia in the Perfused Rat Liver

This study examines the incorporation of 15N from 15NH4Cl into urea and glutamine, predicts the pattern of isotopomers produced as a function of the 15N enrichment of the relevant precursor pools, and presents a means of determining the isotopic enrichment of these pools. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM 15NH4Cl, and the isotopomers of urea and of glutamine produced were determined by gas chromatography-mass spectrometry methodology. Three different nitrogen mass isotopomers of urea were found, containing no, one, or two atoms of 15N. Four glutamine isotopomers were found, containing no 15N, one atom of 15N in either the amino or amide position, or two 15N atoms. A mathematical relationship was deduced that predicts that the relative proportions of the urea isotopomers depends not only on the relative enrichment of 15N in the two precursor pools of urea nitrogen (mitochondrial ammonia and cytoplasmic aspartate) but on their absolute enrichment. This relationship was validated in experiments in which the isotopic enrichment of the substrate, 15NH4Cl, was varied. The proportions of the urea isotopomers produced can be predicted if one knows the 15N enrichment in the two precursor pools. We found that when the 15N enrichment of citrulline and aspartate in the perfusate were used as proxies for that in the mitochondrial ammonia and cytoplasmic aspartate pools we could accurately predict the relative proportion of the three isotopomers. The production of the four nitrogen isotopomers of glutamine could be used to determine the 15N enrichment in the two precursor pools of glutamine nitrogen, the cytoplasmic ammonia and glutamate pools of the perivenous hepatocytes.

Systemic activation of glutamate dehydrogenase increases renal ammoniagenesis: implications for the hyperinsulinism/hyperammonemia syndrome

The hyperinsulism/hyperammonemia (HI/HA) syndrome is caused by glutamate dehydrogenase (GDH) gain-of-function mutations that reduce the inhibition by GTP, consequently increasing the activity of GDH in vivo. The source of the hyperammonemia in the HI/HA syndrome remains unclear. We examined the effect of systemic activation of GDH on ammonia metabolism in the rat. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) is a nonmetabolizable analog of the natural GDH allosteric activator leucine. A dose of 100 mumol BCH/100 g rat resulted in a mild systemic hyperammonemia. Using arterial-venous (A-V) differences, we exclude the liver, intestine, and skeletal muscle as major contributors to this BCH-induced hyperammonemia. However, renal ammonia output increased, as demonstrated by an increase in A-V difference for ammonia across the kidney in BCH-treated animals. Isolated renal cortical tubules incubated with BCH increased the rate of ammoniagenesis from glutamine by 40%. The flux through GDH increased more than twofold when BCH was added to renal mitochondria respiring on glutamine. The flux through glutaminase was not affected by BCH, whereas glutamate-oxaloacetate transaminase flux decreased when normalized to glutaminase flux. These data show that increased renal ammoniagenesis due to activation of GDH can explain the BCH-induced hyperammonemia. These results are discussed in relation to the organ source of the ammonia in the HI/HA syndrome as well as the role of GDH in regulating renal ammoniagenesis.