News Physiol Sci 16: 157-160, 2001;
1548-9213/01 $5.00
News in Physiological Sciences, Vol. 16, No. 4, 157-160,
August 2001
© 2001 Int. Union Physiol. Sci./Am. Physiol. Soc.
The Glutamine/Glutamate Couplet and Cellular Function
Tomas Welbourne1,
Robert Routh2,
Marc Yudkoff3 and
Itzhak Nissim3
1 Departments of Molecular and Cellular Physiology and
2 Molecular and Cellular Pathology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130; and
3 Division of Child Development and Rehabilitation, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4318
 |
Abstract
|
|---|
All cells require glutamine as a nitrogen donor as well as an energy source for cell-specific functions. Understanding how glutamine utilization is metered to these demands is fundamental to basic cell processes as well as to therapeutic manipulation of regulatory mechanisms. The regulatory role of the glutamine/glutamate couplet in cellular function is illustrated for acid-base homeostasis and for production of the extracellular matrix.
|
Introduction
|
|---|
Glutamine (Fig. 1
) is the major source of nitrogen for protein synthesis and is an important oxidizable fuel present in plasma and in culture media. In contrast, glutamate (Fig. 1) is far less prevalent than glutamine, at a plasma concentration only 1/50 of glutamine. This static disparity, however, belies the dynamic relationship of glutamine conversion to glutamate and glutamate's primary role in regulating and fueling cellular processes. The development of this concept and its use in understanding cellular and organ responses to physiological challenges is our thesis.
View larger version (10K):
[in this window]
[in a new window]
|
FIGURE 1. The glutamine/glutamate couplet plus amide-derived NH4+. Dynamic conversion of glutamine to glutamate can be monitored by using uniformly 14C-labeled (•) glutamine or amino-labeled [15N]glutamine (*).
|
|
|
Development of the concept
|
|---|
The conversion of glutamine to glutamate occurs in both the extracellular and intracellular compartments in many cell types and organs (Fig. 1
). These site-specific rates can be monitored by following the formation of glutamate from glutamine either labeled in the amino nitrogen position with 15N (8) or on the carbon skeleton with 14C (6). In proximal tubule-like epithelial cells obtained from pig kidney (LLC-PK1-F+; Fig. 2), the extracellular glutaminase [phosphate-independent glutaminase (PIG)] is localized on the external surface of the plasma membrane in close association with the intrinsic membrane high-affinity glutamate transporters (X–AG subtypes EAAC1 and GLT1; Fig. 2). Although glutamate does not normally accumulate in the media of cells expressing PIG, its contribution to glutamate uptake can readily be demonstrated after the high-affinity glutamate uptake is blocked and following the conversion of uniformly 14C-labeled glutamine to [U-14C]glutamate; extracellular glutamate then accumulates, and this accumulation of radiolabeled glutamate can be eliminated by inhibiting PIG with acivicin (6).
Glutamine is also converted to glutamate by the intracellular phosphate-dependent glutaminase (PDG), whose functional activity is expressed on the cytosolic surface of the inner mitochondrial membrane, as first demonstrated by Kvamme et al. (4). Using isolated mitochondria from pig kidney (Fig. 3), they monitored the appearance of [U-14C]glutamate formed from [U-14C]glutamine in the media and mitochondrial matrix compartments with time and showed that glutamate enrichment in the former preceded and greatly exceeded that of the matrix compartment. Importantly, they also showed that this functional glutaminase was competitively inhibited by glutamate present in the cytosol (12). The presence of the functional glutaminase activity within the cytosolic compartment and inhibited by glutamate, a product of the extracellular glutaminase, suggested that the formation and uptake of extracellular glutamate might regulate the intracellular couplet conversion rate.
View larger version (19K):
[in this window]
[in a new window]
|
FIGURE 3. Evidence for the functional PDG activity at the cytosolic surface of the inner mitochondrial membrane. Intact mitochondria incubated with [14C]glutamine produce [14C]glutamate (label indicated by •), with a far greater specific activity in the cytosol (media) than in the matrix (4). The conversion rate is slowed by increasing the media glutamate concentration, which competes with glutamine (12). If amino-labeled [15N]glutamine (*) is used (20), [15N]glutamate is transaminated with pyruvate to form [15N]alanine (*ALA) and AKG2–. AKG2– is then transported into the mitochondria to support ATP formation. Cytosolic glutamate can be transported via a proton-driven inner membrane carrier (14) into the matrix space and can be deaminated to 15NH4+ and AKG2–.
|
|
Using the proximal tubule-like pig kidney cells, we eliminated extracellular glutamate uptake either by blocking the high-affinity uptake with D-aspartate or by inhibiting PIG with acivicin. Both of these maneuvers produced a 40–50% reduction in the intracellular glutamate content without reducing cellular glutamine content (18). Measurement of the intracellular hydrolysis of [14C]glutamine to [14C]glutamate showed a two- to threefold increase in the conversion rate (18), whereas a tenfold increase in media [15N]glutamate from [2-15N]glutamine under these conditions demonstrated the close proximity of the functional glutaminase to the plasma membrane transporters responsible for the efflux (20). Thus extracellular conversion of glutamine to glutamate coupled to uptake functions as a unit (17) in modulating the intracellular conversion of glutamine to glutamate.
This functional unit can be rendered inoperative by "knocking out" either the EAAC1 or GLT1 glutamate transporter genes in mice. With the EAAC1 gene knockout the intraluminal conversion of glutamine to glutamate results in a large glutamate excretion (11); with the GLT1 gene knockout (16) antiluminal glutamate uptake is reduced, lowering the cytosolic concentration and activating the intracellular glutaminase, resulting in an increased NH4+ excretion (K. Tanaka and T. Welbourne, unpublished observations). Pharmacological knockout of PIG by using acivicin in rats in vivo lowers kidney glutamate content and enhances renal glutamine uptake and NH4+ production (19). These findings are consistent with the functional unit playing a regulatory role in modulating intracellular glutaminase flux in vivo (17).
The fate of glutamate formed intracellularly from glutamine labeled with 15N in the amino position can be studied after removing the functional unit control by using X–AG blockers and/or acivicin. The appearance of [15N]alanine reflects the transamination of [15N]glutamate with pyruvate catalyzed by alanine aminotransferase (ALT), predominantly a cytoplasmic activity, whereas the 15N in NH4+ reflects the deamination of glutamate catalyzed by glutamate dehydrogenase (GDH) present within the matrix space of the mitochondrion (Fig. 2; Refs. 9 and 14). In pig kidney cells, as in most cells, both GDH and ALT pathways are expressed, but the glutamate flux through the cytosolic ALT pathway is normally far higher than the flux through the intramitochondrial GDH pathway (20). However, if the cytosolic pH is reduced by using the acid-loading modality of the X–AG activity (6), cytosolic glutamate is channeled into the mitochondrial GDH pathway via the proton-driven inner membrane glutamate transporter (14). Consequently, the 15N label from glutamate is recovered in 15NH4+ (20). Thus the balance between glutamine's amino nitrogen recovered as alanine versus that recovered as NH4+ provides a window into the prevailing cytosolic acid/base conditions (10, 20).
|
Pivotal role of glutamate formed in the cytosol
|
|---|
The glutamate formed in the cytosol is a pivotal point for dedicated cellular processes, and its fate is determined as depicted in Fig. 4. If glutamate enters the deamination pathway, base and energy (ATP) are produced, as opposed to the transamination pathway, which provides amino acids and energy (ATP) for biosynthetic processes. The cytosolic ALT flux is dependent on the availability of glutamate, pyruvate, and H+ as well as the amount of ALT protein. The mitochondrial GDH flux is dependent on the inner membrane glutamate/H+ transporter (14), H+ and glutamate in the cytosol, and the activity of GDH. Note that the combined flux through both pathways determines the ATP generated from glutamine oxidation, and this in turn is set by the level of the PDG activity. In addition to the cytosolic glutamate concentration, the PDG is dependent on the amount of PDG protein, which in turn is influenced by the acid-base status (1) and growth factors. Hormones and cytokines that upregulate gene expression of key proteins in each pathway, such as glucocorticoids for the catabolic pathway and growth factors for the anabolic pathway, play important and predominantly opposing roles in the adaptive responses of cellular processes detailed below.
View larger version (10K):
[in this window]
[in a new window]
|
FIGURE 4. Cytosolic glutamate metabolism depends on a balance of protons (H+) and hormonal interactions [glucocorticoids (Gc) favor the catabolic pathway, whereas growth factors (GF) favor the anabolic pathway]. The overall flux [the sum of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) fluxes] depends on PDG activity, which is also dependent on protons and growth factors.
|
|
|
Response of glutamine/glutamate to metabolic acidosis
|
|---|
As shown in Fig. 2, in vivo, proximal tubule cells of the kidney respond to a reduction in the body fluid alkaline reserves by increasing glutamine uptake and glutaminase flux coupled specifically to the intramitochondrial GDH pathway. This results in two molecules of NH4+ formed from glutamine and, with complete oxidation of 
-ketoglutarate, two molecules of HCO3–. The amide and amino nitrogens are secreted as NH4+ at the cell's apical surface Na+/H+ (NH4+) exchanger and are excreted in the urine; HCO3– is transported out of the cell at the basolateral Na+-HCO3– cotransporter into the renal vein for a net gain of two molecules of base (Fig. 2). In vitro, pig kidney proximal tubule-like cells also respond to metabolic acidosis with an increased NH4+ production and glutamine conversion to glutamate via the intracellular glutaminase (1, 7). Because the extracellular PIG is HCO3– and glutamine concentration-dependent, lowering both the alkaline reserves and plasma glutamine levels serves as a signal-reducing extracellular glutamine conversion to glutamate, limiting glutamate available for uptake and removing the functional unit's inhibitory influence on the intracellular glutaminase. Simultaneously, the intracellular acidosis drives glutamate into the mitochondrial GDH pathway (9) and thereby reinforces the reduction in cytosolic glutamate (7). The lowered intracellular glutamate content combined with an increased glutamine content (as the result of an adaptive increase in glutamine uptake by an ASC-like transporter; Fig. 2), increases the cytosolic glutamine-to-glutamate ratio and accelerates the PDG flux coupled to the GDH-base generating pathway (9). By regulating the extracellular couplet according to the plasma HCO3– concentration, base generation can be metered to deficits in systemic alkaline reserves and the cellular acidosis can channel the glutamate formed from PDG into the base generation GDH pathway. Because the functional unit's control of the PDG is sensitive to reduced HCO3– concentration and not reduced pH (7), whereas the cytosolic glutamate content is pH sensitive 8), these two signals would operate together in metabolic acidosis and in opposition in respiratory acidosis, thereby providing the flexibility required to maintain acid-base homeostasis under the threat of either metabolic or respiratory disturbances.
|
Response of glutamine/glutamate to troglitazone
|
|---|
Mesangial and proximal tubule cells elaborate an extracellular matrix, the overexpression of which during inflammatory diseases results in glomerulosclerosis and interstitial nephritis (15). The synthesis of extracellular matrix proteins, e.g., collagen and laminin, requires an adequate supply of precursor amino acids dependent in large part on glutamine's amino nitrogen and the transamination pathways coupled to protein synthesis (Fig. 2). The thiazolidinedione troglitazone has been shown to halt mesangium expansion in animal models of type 2 diabetes (5) and to act directly on rat mesangial cells in reducing collagen type I and laminin synthesis (13). Since the glutamine/glutamate couplet shows regulatory properties, we asked whether the effect of troglitazone on matrix and, specifically, laminin synthesis might be mediated through the couplet. To test this, the sum of 15NH4+ and [15N]alanine formed from amino-labeled [15N]glutamine was taken as an index of the glutaminase flux (Fig. 2). The flux through the ALT/GDH pathways was determined from the [15N]alanine and 15NH4+ fluxes, respectively, in pig kidney cells incubated in the presence or absence of 20 µM troglitazone. As shown in Fig. 5A, the predominant glutamate flux is normally through the biosynthetic transamination pathway (see Fig. 2). However, in the presence of troglitazone the transamination flux is sharply curtailed, and this reduction is paralleled by a proportional drop in the assayable ALT activity (Fig. 5B). The 15NH4+ flux from labeled glutamate is increased, shifting the pivotal glutamate flux balance far toward the GDH flux (Fig. 4). Since this NH4+ is formed from labeled glutamate derived from glutamine within the cytosolic compartment, one may infer that a drop in cytosolic pH has occurred as the result of troglitazone exposure since the assayable GDH activity did not increase (Fig. 5B). Furthermore, the overall glutaminase flux (the sum of 15NH4+ and [15N]alanine) decreases, indicating a reduction in the glutamine conversion to glutamate (Fig. 2). The measured intracellular glutamate concentration increases by 26%, which is consistent with glutamate from the inhibited ALT pathway backing up in the cytosol and slowing the PDG flux. This in turn limits the ATP available from the oxidation of glutamate's carbon skeleton (Fig. 2) (14). These responses allocate the cells' nitrogen resources away from the biosynthetic pathway and into the base generating pathway, as reflected by the inverse relationship between 15NH4+ production (Fig. 5A) and cellular laminin content (Fig. 5C).Thus the regulatory role of glutamate in the functional glutaminase flux can be demonstrated both in the antegrade direction in response to acid-base disturbances and in the retrograde direction in the response to troglitazone.
View larger version (28K):
[in this window]
[in a new window]
|
FIGURE 5. Troglitazone's action on the glutamine/glutamate couplet and cellular responses. A: shift of cytosolic glutamate derived from [15N]glutamine (99 atom %excess) from alanine (ALA) synthesis into NH4+ formation in LLC-PK1-F+ monolayers incubated in DMEM or DMEM plus 20 µM troglitazone for 16 h. Overall PDG flux as the sum of [15N]ALA + 15NH4+ was decreased by 16% (774 ± 9 to 654 ± 11 nmol/mg; P < 0.01). Monolayer glutamate content increased from 78 ± 8 to 98 ± 3 nmol/mg protein (P < 0.03). Results are means ± SE; n = 3. B: assayable GDH and ALT activity in the above monolayers of LLC-PK1-F+ after incubation for 16 h under the above conditions. C: laminin content of LLC-PK1-F+ cells after incubation under the above conditions. Laminin content was assayed in 50- µg protein aliquots of cell lysates as previously described (13).
|
|
|
Perspectives
|
|---|
The glutamine/glutamate couplet offers a novel perspective on how growth factors such as transforming growth factor (TGF)-ß regulate the extracellular matrix expansion (in addition to their well-established effects on matrix protein gene expression; Ref. 15). For example, TGF-ß may upregulate the PDG and Na+/H+ exchanger 3 (NHE3) gene expression and downregulate the PIG gene expression so that the intracellular glutaminase is accelerated with the glutamate that is formed coupled to the transamination pathway. Of course, increasing matrix protein gene expression is necessary for matrix expansion, but synthesis of matrix protein will not proceed unless there are adequate precursor amino acids made available via the glutamine/glutamate couplet. Thus it is reasonable that effects on PDG expression and transamination pathways should accompany these recognized actions of TGF-ß.
Troglitazone has been shown to interdict extracellular matrix expansion in experimental diabetes associated with a decrease in levels of TGF-ß and extracellular matrix protein mRNA (2). However, by inhibiting the transamination pathway, shifting the nitrogen into the catabolic GDH pathway, and at the same time slowing the PDG flux, troglitazone effectively "starves" the extracellular matrix expansion of precursors. Mechanistically, this shift in glutamate's fate can be mimicked by lowering the cytosolic pH, suggesting that one of troglitazone's actions is to impair the cell's ability to extrude protons. Possibly, troglitazone blocks activation of protein kinase C (PKC) by increasing diacylglycerol (DAG) kinase activity (2) and limiting available DAG. This could prevent PKC activation of the NHE3 (3), and thereby the cell retains protons in the cytosol, resulting in the shift of glutamate into the catabolic pathway. Together these effects would limit the glutamine/glutamate couplet's flow of nitrogen and energy required for extracellular matrix synthesis. Hopefully these studies illustrate the basis of how cellular metabolism and physiological function are integrated on the molecular level and in so doing may reveal more about pathophysiological processes.
|
Acknowledgments
|
|---|
We thank Dr. Neil Granger for reading the manuscript and for helpful comments.
Research in the laboratory of T. Welbourne is supported by the Southern Arizona Foundation and Genentech. R. Routh is a recipient of a Research Fellowship from the Louisiana Affiliate of the American Heart Association. I. Nissim is supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-53761) .
|
References
|
|---|
-
Gstraunthaler G, Holcomb T, Feifel E, Liu W, Spitaler N, and Curthoys N. Differential expression and acid-base regulation of glutaminase mRNAs in gluconeogenic LLC-PK1-FBPase+ cells. Am J Physiol Renal Physiol 278: F227–F237, 2000.[Abstract/Free Full Text]
-
Isshiki K, Haneda M, Koya D, Maeda S, Sugimoto T, and Kikkawa R. Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats. Diabetes 49: 1022–1032, 2000.[Abstract]
-
Karim ZG, Chambrey R, Chalumeau C, Defontaine N, Warnock DG, Pallard M, and Poggioli J. Regulation by PKC isoforms of Na+/H+ exchanger in luminal membrane vesicles isolated from cortical tubules. Am J Physiol Renal Physiol 277: F773–F778, 1999.[Abstract/Free Full Text]
-
Kvamme E, Torgner IA, and Roberg B. Evidence indicating that pig renal phosphate dependent glutaminase has a functionally predominant external localization in the inner mitochondrial membrane. J Biol Chem 266: 12185–12192, 1991.[Abstract/Free Full Text]
-
McCarthy KJ, Routh RE, Shaw W, Walsh K, Welbourne TC, and Johnson JH. Troglitazone halts diabetic glomerulosclerosis by blockade of mesangial expansion. Kidney Int 58: 2341–2350, 2000.[ISI][Medline]
-
Meade D, Chess C, and Welbourne TC. Glutamate transport and cellular glutamine metabolism: regulation in LLC-PK1 and LLC-PK1-F+ cell lines. Am J Physiol Cell Physiol 274: C1616–C1624, 1998.[Abstract/Free Full Text]
-
Mu X and Welbourne T. Response of LLC-PK1_F+ cells to metabolic acidosis. Am J Physiol Cell Physiol 270: C920–C925, 1996.[Abstract/Free Full Text]
-
Nissim I. Newer aspects of glutamine/glutamate metabolism: role of acute pH changes. Am J Physiol Renal Physiol 277: F493–F497, 1999.[Abstract/Free Full Text]
-
Nissim I, Sahai A, Sandler R, and Tannen RL. The intensity of acidosis differentially alters the pathways of ammoniagenesis in LLC-PK1 cells. Kidney Int 45:1014–1019, 1994.[ISI][Medline]
-
Nissim I, States B, Nissim I, Lin ZP, and Yudkoff M. Hormonal regulation of glutamine metabolism by OK cells. Kidney Int 47: 96–105, 1995.[ISI][Medline]
-
Peghini P, Jaryen J, and Stoffel W. Glutamate transporter EAAC1 deficient mice develop dicarboxylic acid aminoaciduria and behavioral abnormalities but no neurodegeneration. EMBO J 16: 3822–3832, 1997.[ISI][Medline]
-
Roberg B, Torgner IA, Laake J, Takumi Y, Otterson OP, and Kvamme E. Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase. Am J Physiol Cell Physiol 279: C648–C657, 2000.[Abstract/Free Full Text]
-
Routh ER, Johnson JH, and McCarthy KJ. Direct action of troglitazone on mesangial cell behavior: troglitazone suppresses the secretion of type 1 collagen by mesangial cells in vitro. Kidney Int. In press.
-
Schoolwerth AC and LaNoue KF. Transport of metabolic substrates in renal mitochondria. Annu Rev Physiol 47: 143–171, 1985.[ISI][Medline]
-
Sharma K and Ziyadeh FN. Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-ß as a key mediator. Diabetes 44: 1139–1146, 1995.[Abstract]
-
Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Taksunobu K, Iwama H, Nishikawa T, Ichara N, Kikuchi T, Okuyama S, Kawashima N, Hori S, Takimoto M, and Wada K. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276: 1699–1702, 1997.[Abstract/Free Full Text]
-
Welbourne TC and Matthews JC. Glutamate transport and renal function. Am J Physiol Renal Physiol 277: F501–F505, 1999.[Abstract/Free Full Text]
-
Welbourne TC and Mu X. Extracellular glutamate flux regulates intracellular glutaminase activity in LLC-PK1-F+ cells. Am J Physiol Cell Physiol 268: C1418–C1424, 1995.[Abstract/Free Full Text]
-
Welbourne T, Mu X, and Evans S. Enteral glutamine modulates renal glutamine utilization. J Nutr 126: 1137S–1141S, 1996.
-
Welbourne T and Nissim I. Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with 15N. Am J Physiol Cell Physiol 280: C1151–C1159, 2001.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
M. Andratsch, E. Feifel, L. Taylor, M. O'Hayre, H. Schramek, N. P. Curthoys, and G. Gstraunthaler
TGF-beta signaling and its effect on glutaminase expression in LLC-PK1-FBPase+ cells
Am J Physiol Renal Physiol,
September 1, 2007;
293(3):
F846 - F853.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Welbourne, E. Friday, R. Fowler, F. Turturro, and I. Nissim
Troglitazone acts by PPAR{gamma} and PPAR{gamma}-independent pathways on LLC-PK1-F+ acid-base metabolism
Am J Physiol Renal Physiol,
January 1, 2004;
286(1):
F100 - F110.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. L. O'Malley, Y.-J. I. Jong, Y. Gonchar, A. Burkhalter, and C. Romano
Activation of Metabotropic Glutamate Receptor mGlu5 on Nuclear Membranes Mediates Intranuclear Ca2+ Changes in Heterologous Cell Types and Neurons
J. Biol. Chem.,
July 18, 2003;
278(30):
28210 - 28219.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Coates, I. Nissim, H. Battarbee, and T. Welbourne
Glitazones regulate glutamine metabolism by inducing a cellular acidosis in MDCK cells
Am J Physiol Endocrinol Metab,
October 1, 2002;
283(4):
E729 - E737.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Welbourne, G. Su, G. Coates, R. Routh, K. McCarthy, and H. Battarbee
Troglitazone induces a cellular acidosis by inhibiting acid extrusion in cultured rat mesangial cells
Am J Physiol Regulatory Integrative Comp Physiol,
June 1, 2002;
282(6):
R1600 - R1607.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Routh, K. McCarthy, and T. Welbourne
Troglitazone inhibits glutamine metabolism in rat mesangial cells
Am J Physiol Endocrinol Metab,
January 1, 2002;
282(1):
E231 - E238.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Introduction
Development of the concept
-glutamyltranspeptidase-glutaminase reaction (Y), with labeled glutamate measured in the media after uptake by X–AG is blocked (