Department of Cellular and Molecular Physiology, Louisiana State
University Medical Center in Shreveport, Shreveport, Louisiana
71130-3932
The glutamate (Glu) transporter may modulate cellular glutamine
(Gln) metabolism by regulating both the rates of hydrolysis and
subsequent conversion of Glu to
-ketoglutarate and
NH+4. By delivering Glu, a competitive
inhibitor of Gln for the phosphate-dependent glutaminase (PDG) as well
as an acid-load activator of glutamate dehydrogenase (GDH) flux, the
transporter may effectively substitute extracellularly generated Glu
from the
-glutamyltransferase for that derived intracellularly from
Gln. We tested this hypothesis in two closely related porcine kidney
cell lines, LLC-PK1 and LLC-PK1-F+,
the latter selected to grow in the absence of glucose, relying on Gln
as their sole energy source. Both cell lines exhibited PDG suppression
as the result of Glu uptake while disrupting the extracellular
L-Glu uptake, with
D-aspartate-accelerated
intracellular Glu formation coupled primarily to the ammoniagenic
pathway (GDH). Conversely, enhancing the extracellular Glu formation
with p-aminohippurate and Glu uptake
suppressed intracellular Gln hydrolysis while
NH+4 formation from Glu increased. Thus these
results are consistent with the transporter's dual role in modulating
both PDG and GDH flux. Interestingly, PDG flux was actually higher in
the Gln-adapted LLC-PK1-F+
cell line because of a two- to threefold enhancement in Gln uptake despite greater Glu uptake than in the parental
LLC-PK1 cells, revealing the
importance of both Glu and Gln transport in the modulation of PDG flux.
Nevertheless, when studied at physiological Gln concentration, PDG flux
falls under tight Glu transporter control as Gln uptake decreases,
suggesting that cellular Gln metabolism may indeed be under Glu
transporter control in vivo.
glutamine uptake; ammonium formation; phosphate-dependent
glutaminase; glutamate dehydrogenase;
-glutamyltransferase
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INTRODUCTION |
GLUTAMATE (Glu) transporter activity potentially
regulates two key pathways of cellular glutamine (Gln) metabolism as
illustrated in Fig. 1. By maintaining
cellular Glu concentration (32), which in turn acts as a competitive
inhibitor of phosphate-dependent glutaminase (PDG; Fig. 1,
reaction 1
) (10, 17, 27), the transporter suppresses intracellular Glu formation. On the other hand,
the Glu transporter subtype EAAC-1 delivers an acid load (15), and we
have shown by Northern blot analysis that this transporter mRNA is
expressed in both the
LLC-PK1-F+
and parental LLC-PK1 cell lines
(unpublished observations). Thus, by lowering cell pH, Glu transporter
activity potentially accelerates Glu flux through the glutamate
dehydrogenase (GDH) pathway (Fig. 1, reaction
4+) (19, 21). Accordingly, transporter activity may suppress PDG while accelerating GDH, effectively substituting extracellular Glu while sparing Gln as a fuel.

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Fig. 1.
Glu transporter regulation of intracellular glutaminase
(reaction 1) and glutamate
dehydrogenase flux (reaction 4). Glu
generated extracellularly from Gln via -glutamyltransferase
(reaction 2) is transported into
cell maintaining high levels of intracellular Glu, a competitive
inhibitor of Gln at reaction 1. Net
acid transport (H+) associated
with Glu accelerates flux through reaction
4, generating NH+4. Ala
formation resulting from alanine aminotransferase activity
(reaction 5) and Gln transamination
(reaction 3) are not directly
affected by Glu transporter activity. Putative Gln transport
(reactions 6 and
7) may reflect an exchange reaction
of Gln for Ala as well as
Na+-dependent uptake coupled to
reaction 1. -KG,
-ketoglutarate; + and , accelerated and inhibited fluxes,
respectively.
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As depicted in Fig. 1, the rate of Glu transport into the cell is
dependent on the available extracellular Glu, either that preformed in
the medium or that generated by
-glutamyltransferase (
-GT) from
extracellular Gln (Fig. 1, reaction
2). Because the preformed Glu, at a physiological
concentration, i.e., 10-30 µM (3), is inadequate to sustain Glu
transport, continuous extracellular formation via
-GT is required.
Consequently, cells expressing a higher
-GT might be expected to
exhibit a greater Glu uptake and tighter regulatory control.
To test this, two related cell lines with very different
-GT
activities were studied. Gstraunthaler and Handler (12) derived the
LLC-PK1-F+
cell line from the parental
LLC-PK1 cell line by selection for growth in a glucose-free medium containing
L-Gln. Noteworthy, the
LLC-PK1-F+
cells express a twofold higher
-GT activity (11, 12) than does the
parental cell line, suggesting that extracellular Glu availability
should be greater, resulting in a higher Glu transporter flux and
reduced Gln utilization (Fig. 1). This of course would be paradoxical,
since the
LLC-PK1-F+
cells were selected for growth with Gln as the sole fuel. Because Glu
acts as a competitive inhibitor, the cellular Gln concentration must
also be considered when evaluating the flux through PDG (Fig. 1,
reaction 1+). In this regard, little
is known about the Gln transport systems in the
LLC-PK1-F+
cell line, although systems A, ASC, and L are present in the parental
cell line (24, 26). Nevertheless, if Gln uptake was also increased, so
that cellular Gln concentration rose proportionally or even more than
the Glu concentration in the
LLC-PK1-F+
cell line, then PDG flux may indeed be maintained at least equal to, if
not greater, than that in the parental cell lines. Indeed, the results
to follow are consistent with a higher glutaminase flux in the
Gln-adapted
LLC-PK1-F+
cell as the result of upregulated Gln transport; in contrast the PDG
flux in the LLC-PK1 cell line
appeared more suppressed. To test this,
D-aspartate (Asp) or
p-aminohippurate (PAH), the latter an
activator of
-GT, (30), was used to block or enhance extracellular
L-Glu uptake and, if the dual
role hypothesis is correct, increase
(D-Asp) or decrease (PAH)
intracellular Glu formation from PDG while accelerating ammonium
(NH+4) formation from GDH. In line with this,
the results to follow show that cellular Glu concentration and
glutaminase flux responded to modulation of extracellular Glu
uptake while flux through the GDH pathway accelerated with transporter
turnover, results consistent with a dual role for the Glu transporter
in regulating a major pathway of cellular energy production.
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MATERIALS AND METHODS |
LLC-PK1-F+
cells (12) and LLC-PK1 cells
obtained from the American Type Culture Collection were grown to
confluence on 60-mm plastic dishes in DMEM containing (in mM) 1.8 L-Gln, 0.034 L-Glu, 28 HCO
3, and either 5 or 20 D-glucose (5 for F+ cells) as well as 10% fetal
bovine serum (Hyclone, Ogden, UT) in 5%
CO2-95% atmosphere. Experiments
were performed 4-8 days after seeding with fresh medium exchanged
daily. Seed plates are routinely screened for
Mycoplasma with contamination
monitored by use of a Mycoplasma PCR
primer kit (Stratagene Cloning Systems, La Jolla, CA).
Experimental design.
These experiments were designed to characterize each component of the
-GT-Glu transporter unit depicted in Fig.
1. The two components, extrinsic plasma
membrane
-GT activity and the intrinsic membrane Glu transporter,
were separately assessed under physiological conditions, i.e., medium
Glu and Gln concentration at 0.034 and 0.9 mM, respectively. The
balance between extracellular Glu formation and uptake was then
determined from the medium Glu content after 45 min of incubation in
DMEM. To confirm that Gln conversion to Glu indeed occurs
extracellularly and that this conversion is an expression of
-GT
activity, formation of
[14C]Glu from
[14C]Gln was measured
in the absence and presence of the
-GT inhibitor, acivicin (AT-125)
(16). Monolayers were presented with DMEM containing 0.9 mM
L-Gln and tracer amounts of
radiolabeled
[U-14C]Gln (2 µCi/ml) for 1 and 2.5 min with breakdown rates determined in the
presence of Glu transport blockers,
D-Asp (5 mM) and
DL-threo-
-hydroxyaspartate (THA, 0.5 mM), which are potent inhibitors of the high-affinity Glu
transporters (1, 2). The medium was then removed and exchanged for the
same medium minus the tracer plus 0.75 mM AT-125 for 15 min, after
which the tracer-containing medium plus AT-125 was exchanged for a
second determination of the 1- and 2.5- min rates. The rate of
extracellular Gln conversion to Glu was then estimated from the counts
per minute in Glu divided by the Gln specific activity (cpm/nmol; rates
expressed in nmol Gln converted to
Glu · min
1 · mg
protein
1). Simultaneously
measured Glu accumulation, determined from the increment in medium Glu
concentration times volume (2.5 ml), provided a comparison between
-GT-generated Glu and that accumulating via efflux from the cells.
To determine the Glu transporter activity under physiological
conditions, monolayers were incubated with DMEM containing 0.034 mM Glu
plus tracer amounts of
[U-14C]Glu (2 µCi/ml, 158 mCi/mmol, Sigma, St. Louis, MO). After 1 min, the
radioactive medium was suctioned off, and dishes were placed on ice and
repeatedly (6 times) washed with ice-cold PBS (pH 7.4). Ice-cold 5%
TCA (1 ml) was added to the cells scraped into tubes and homogenized
with a Polytron (half-speed for 20 s). After transfer of to 1.5-ml
Microfuge tubes, the homogenates were centrifuged (10,000 g for 10 min), the supernatants were retained for analysis of radioactive Glu, and the pellets were processed for protein determination by the dye binding assay with BSA
as the standard (4). One-minute Glu uptake rate was determined from the
monolayer radioactive Glu (cpm) divided by the medium Glu specific
activity (rates expressed in
nmol · min
1 · mg
protein
1). Radiolabeled
Glu accounted for 92 ± 4% of the total cellular radioactivity
after 1 min.
To assess the role of extracellular Glu formation on intracellular Gln
conversion to Glu and flux through the GDH pathway, 1 mM PAH was added
to DMEM containing 0.9 mM L-Gln
over 45 min. PAH increases the maximal velocity of
-GT-catalyzed
hydrolysis of Gln (30). To confirm that indeed more Glu was being
formed extracellularly, parallel experiments were carried out in which the Glu transport step was blocked with the rise in medium Glu accumulating in the presence of PAH taken as the enhancement of
-GT-Glu production. To confirm that more Glu entered the cells in
the presence of PAH, the medium and cellular Glu contents were measured
after 45 min. A demonstrable increase in extracellular Glu accumulation
with Glu uptake blocked and the rise in cellular Glu without
extracellular accumulation were then taken to indicate an increased
flux of extracellular Glu through the
-GT-Glu transporter unit.
To assess the effect of modulating Glu delivery on intracellular Gln
and Glu metabolism, estimates of PDG and GDH fluxes were made.
Intracellular Gln-to-Glu conversion rate was determined as an index of
PDG flux as previously described (32). Briefly, the monolayers were
first incubated for 45 min at 37°C in DMEM containing either 1.8 or
0.9 mM L-Gln, after which
samples were taken, and the medium was replaced with fresh DMEM
containing radiolabeled
[U-14C]Gln (2 µCi/ml, 250 mCi/mmol, NEN, Boston, MA) plus Glu transport blockers
(see above) to prevent uptake of extracellularly formed Glu. After 1 min of incubation (37°C, 5%
CO2), the plates were processed
as above, and the supernatant was fractioned by HPLC to isolate
radioactive Glu and Gln (32). The rate of intracellular Glu formation
from Gln was then determined from monolayer radioactive Glu (cpm)
divided by medium Gln specific activity (rates expressed in
nmol · min
1 · mg
protein
1) and taken as an
estimate of flux through the PDG (Fig. 1, reaction 1). The rate of Gln uptake (expressed in
nmol · min
1 · mg
protein
1) was also
determined in these experiments (32). Note that unidirectional Gln
transport into the cell measured at 1 min exceeded the intracellular conversion to Glu by at least a factor of 3 in both the cell lines; consequently the transport step is not rate limiting for the
intracellular conversion. In addition, >82% of the monolayer
radioactivity could be recovered in combined Gln plus Glu peaks at 1.0 min, consistent with limited conversion of radiolabeled Glu over this
time course; a longer time course would, of course, reflect the
contribution of downstream reactions rather than the initial
glutaminase flux. Although the intracellular conversion rates obtained
are only an estimate of the intracellular glutaminase flux, they do
provide a comparison of the same estimated flux between the two cell
lines (under similar conditions) as well as within the same cell line (before and after lowering or raising cellular Glu).
We previously utilized D-Asp (10 mM for 18 h) to block L-Glu
uptake and lower intracellular Glu (32). In the present acute 45-min
study, D-Asp (5 mM) was used to
both block L-Glu uptake and to
deliver an acid load. To confirm that
D-Asp effectively blocks the
uptake, radiolabeled
L-[U-14C]Glu
(2 µCi/ml) was added to the DMEM and disappearance of the labeled
L-Glu was determined after 45 min. In the presence of D-Asp,
97 ± 2 and 95 ± 4% of the radiolabeled L-Glu
remained in the medium in the
LLC-PK1-F+
and LLC-PK1 cell lines,
respectively; control monolayers, on the other hand, removed 51 ± 2 and 60 ± 4 of the radiolabeled L-Glu, respectively
(n = 3 pairs for each line). These
results are in agreement with our previous study showing >95%
inhibition of
L-[U-14C]Glu
uptake by 10 mM D-Asp (32). To
confirm that 5 mM D-Asp would
lower cell pH as well as block Glu uptake under these conditions, monolayers were grown to confluence in especially designed chambers and
loaded with 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF, Molecular Probes, Eugene OR) by exposing them to 10 µM of the ester
BCECF-AM for 15 min in HEPES-buffered DMEM minus phenol red and FCS.
The chamber was then viewed with an epifluoroscope (Olympus IMT-2)
equipped with a fluorescence detector (Photon Technology Instrument 710 PMT), and the fluorescence was measured at 440 and 495 nm with a 530-nm
emission wavelength. The
high-K+-nigericin technique,
essentially as described by Thomas et al. (29), was used to calibrate
cell pH. Estimated cell pH was 7.55 ± 0.09 in HEPES-buffered DMEM
at 35°C and promptly decreased 0.23 ± 0.08 units with exchange
of the DMEM to one with 5 mM
D-Asp medium; this decline in
estimated cell pH was maintained for at least 15 min. Thus the Glu
transporter's hypothesized dual role in modulating the glutaminase and
GDH fluxes was tested, since both the block of
L-Glu uptake and decrease in
cellular pH were demonstrated.
To assess flux through GDH vs. transamination
(reaction 4 vs.
5), NH+4
and alanine (Ala) formation were measured in the absence and presence
of D-Asp or PAH. The
steady-state uptake of Gln and formation of
NH+4, Ala, and Glu were determined from
changes in the metabolite concentration over the 45-min incubation
times medium volume (3 ml); medium incubated in the absence of
monolayers served as a blank (rates, corrected for spontaneous
breakdown, expressed in
nmol · min
1 · mg
protein
1). A shift in the
NH+4-to-Ala formation ratio was taken as
evidence consistent with a fall in cell pH.
Analyses.
Medium and monolayer Glu, Gln, Ala, and Asp concentrations were
determined on TCA extracts as previously described (32). Briefly, amino
acids in the supernatants were derivatized with o-phthaldialdehyde, separated by HPLC,
and quantitated with a fluorometric detector; retention times for Asp,
Glu, Gln, and Ala were 5.0, 7.8, 11.3, and 16.0 min, respectively.
Samples were spiked with homoserine as an internal standard. Recoveries
of stock radiolabeled
L-[14C]Glu
and
L-[14C]Gln
added to the column were 95 ± 6 and 87 ± 5%, respectively (n = 3).
NH+4 concentration was measured by the microdiffusion method (32) and protein by dye binding (4) with BSA as a
standard.
-GT activity was assayed in situ as described (19). Medium
HCO
3 concentration was routinely measured at the termination of these 45-min experiments as a check on
spontaneous acidosis, which could influence the metabolic pathways (19,
21). However, over this 45-min interval, there was little change in
HCO
3 concentration (27.3 ± 0.3 and 27.4 ± 0.3 vs. initial 28 mM for
LLC-PK1-F+
and LLC-PK1 monolayers,
respectively), which would not be expected to affect Gln utilization
under these conditions.
Statistical comparisons were made between the two cell lines with the
unpaired Student's t-test or between
untreated monolayers and those exposed to
D-Asp or PAH for 45 min with the
paired Student's t-test; for multiple
group comparisons, control, AT-125, and
D-Asp, ANOVA (repeated
measurements) and a corrected t-test
(Bonferroni) were used. When directional changes were predicted based
on the a priori hypothesis (Fig. 1), a one-tailed
t table was employed; otherwise a
two-tailed t table was consulted.
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RESULTS |
The Glu content measured after 45 min of incubation in medium
containing 1.8 mM L-Gln and 34 µM L-Glu is shown in Fig.
2 (control). In the
LLC-PK1-F+
cells, Glu accumulated in excess of that preformed at the rate of 0.52 ± 0.18 nmol · min
1 · mg
protein
1, which contrasted
with a deficit in medium Glu content observed in the
LLC-PK1 cell line (
0.91 ± 0.07 nmol · min
1 · mg
protein
1,
P < 0.001). The Glu transporter
activity measured over 1 min at the preformed medium Glu concentration
(34 µM) was not different in the two cell lines (1.08 ± 0.13 and
1.38 ± 0.08 nmol · min
1 · mg
protein
1 for
LLC-PK1-F+
and LLC-PK1, respectively; see
MATERIALS AND METHODS for details). Therefore the accumulation as opposed to deficit in the medium Glu
content cannot be explained by a difference in transporter activity
operating on the preformed medium Glu. On the other hand,
-GT
activity was very different in the two cell lines (51 ± 6 and 28 ± 5 nmol · min
1 · mg
protein
1) when assayed in
situ with the artificial substrate
-glutamyl-p-nitroanalide (see
MATERIALS AND METHODS) and in
confirmation of the originally observed difference between the two cell
lines when assayed in homogenates (11, 12). In support of the medium
Glu differences being attributable to the different
-GT activity,
AT-125 was added to the medium and Glu content was determined after 45 min;
-GT activity measured after 45 min was reduced >90% in both
cell lines (3 ± 3 and 2 ± 3 nmol · min
1 · mg
protein
1 for the
LLC-PK1-F+
and LLC-PK1, respectively). With
almost complete elimination of
-GT activity in the
LLC-PK1-F+
cells (Fig. 2, AT-125), medium Glu accumulation reversed to uptake (0.52 ± 0.18 to
0.41 ± 0.08 nmol · min
1 · mg
1,
control vs. AT-125, P < 0.001) and further exacerbated the Glu deficit already existing in the
medium in the LLC-PK1 cell line (
0.91 ± 0.07 to
1.26 ± 0.06 nmol · min
1 · mg
1,
P < 0.01).

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Fig. 2.
Medium Glu content measured after 45-min incubation of
LLC-PK1-F+
(n = 15) and
LLC-PK1
(n = 9) cells in DMEM in absence and
presence of 0.75 mM AT-125. Results are means ± SE with differences
between
LLC-PK1-F+
and LLC-PK1 monolayers determined
by unpaired 1-tailed t-test;
differences between control and AT-125-treated monolayers determined by
paired 1-tailed t-test.
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To confirm that extracellular Glu was indeed formed from Gln via
-GT
activity, the initial rate of extracellular radiolabeled L-[14C]Gln
conversion to
L-[14C]Glu
was monitored in the absence and presence of AT-125; in addition, 5 mM
D-Asp and 0.5 mM THA were
present to prevent uptake of the extracellularly generated
L-[14C]Glu
(see MATERIALS AND METHODS,
Experimental design). As shown in
Fig. 3, Gln is really broken down to Glu
extracellularly, which accumulates under these conditions at a
surprisingly high rate (2.1 ± 0.4 and 1.6 ± 0.2 nmol · min
1 · mg
protein
1 at 1 and 2.5 min).
In the presence of AT-125, this extracellular conversion was virtually
eliminated, falling to only 15 and 8% of the control rates (0.31 ± 0.32 and 0.12 ± 20 nmol · min
1 · mg
protein
1 at 1 and 2.5 min).
Furthermore Glu accumulated in the medium at a rate equal to the
extracellular hydrolysis rate at 1 min (2.9 ± 0.6 vs. 2.1 ± 0.4 nmol · min
1 · mg
protein
1, respectively) and
significantly higher at 2.5 min (3.5 ± 0.6 vs. 1.6 ± 0.2 nmol · min
1 · mg
protein
1,
P < 0.05). In the AT-125-treated
monolayers, Glu accumulation far exceeded the negligible extracellular
formation rate at 2.5 min (1.5 ± 0.3 vs. 0.12 ± 0.20 nmol · min
1 · mg
protein
1), demonstrating
cellular Glu efflux in the presence of
D-Asp as previously shown in
Glu-loaded membrane vesicles (8, 28). Collectively, these results show
that extracellular Glu generated from Gln via
-GT (Fig. 1) provides
substrate for the Glu transporter and that extracellular Glu may
reflect de novo formation or transporter efflux or both.

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Fig. 3.
Extracellular [14C]Gln
conversion to [14C]Glu
in control and AT-125-treated monolayers measured at 1 and 2.5 min
under conditions of transport block (5 mM
D-Asp). Results are means ± SE from 3 experiments. * Statistical difference,
P < 0.01.
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The role that
-GT and Glu transporter operating as a unit play in
maintaining the cellular Glu content in these two cell lines is shown
in Fig. 4. In the
LLC-PK1-F+
cells, Glu content was significantly higher than in the
LLC-PK1 cells (175 ± 6 vs. 131 ± 33 nmol/mg, P < 0.05) in line with their greater
-GT activity and Glu transport.
With the extracellular source of Glu eliminated, either by inhibition
of
-GT with AT-125 or by blocking of
L-Glu uptake with 5 mM
D-Asp, cellular Glu decreased 13 and 32%, respectively, in the
LLC-PK1-F+
cell line; the same maneuvers reduced cellular Glu 16 and 42% in the
LLC-PK1 cell line, demonstrating
that extracellular formation and transport into both cell lines
maintains cellular Glu concentration, as depicted in Fig. 1. Because
L-Glu competes with Gln in
inhibiting PDG (10, 27), the cellular
L-Gln content was measured in
these cells and shown in Fig. 5.
Surprisingly, the Gln-adapted
LLC-PK1-F+
cell line showed a nearly threefold higher Gln content compared with
the LLC-PK1 cell line (140 ± 4 vs. 50 ± 19 nmol/mg protein, P < 0.001). Furthermore, the rate at which Gln was taken up into the cells
was measured over 1 min by using
L-[14C]Gln
(see MATERIALS AND METHODS) and was
determined to be 17 ± 1 and 6 ± 2 nmol/mg protein for the
LLC-PK1-F+
and LLC-PK1 cells, respectively
(P < 0.01). Thus the threefold greater Gln transport rate under these conditions appears to maintain the higher cellular Gln concentration in the
LLC-PK1-F+
cell line. Neither AT-125 nor
D-Asp significantly affected the Gln content of either cell line. Consequently, the ratio of the glutaminase inhibitor L-Glu to
the natural substrate L-Gln is actually highest in the LLC-PK1
cells (2.6 vs. 1.25 in the
LLC-PK1-F+
cells) and reduced with D-Asp to
2.0 and 1.03 in the two cell lines, respectively, entirely as the
result of the fall in L-Glu.

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Fig. 4.
Monolayer Glu content determined after 45-min incubation in 1.8 mM
L-Gln DMEM, DMEM + 0.75 mM
AT-125, or DMEM + 5 mM D-ASP.
Results are means ± SE from 15 and 8 plates for each group for
LLC-PK1-F+
and LLC-PK1 cells, respectively,
with statistical differences detected by ANOVA and a corrected
(Bonferroni) t-test.
* P < 0.05.
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Fig. 5.
Monolayer Gln content measured after 45-min incubation in 1.8 mM
L-Gln DMEM, DMEM + 0.75 AT-125,
or DMEM + 5 mM D-ASP. Results
are means ± SE from 15 and 8 plates for each group for
LLC-PK1-F+
and LLC-PK1 cells, respectively.
* P < 0.001.
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In line with the inhibitor-to-substrate ratio, the estimated
intracellular glutaminase flux (Fig. 6) was
significantly lower in the LLC-PK1
compared with
LLC-PK1-F+
cells (1.7 ± 0.4 vs. 4.2 ± 0.7 nmol · min
1 · mg
protein
1,
P < 0.05).
D-Asp accelerated this estimated
glutaminase flux 47% in the
LLC-PK1-F+
cells (4.2 ± 0.7 to 6.1 ± 1.1 nmol · min
1 · mg
protein
1,
P < 0.05) and 118% in the
LLC-PK1 cells (1.7 ± 0.4 to
3.7 ± 0.5 nmol · min
1 · mg
1,
P < 0.05). These results show that
the glutaminase flux is under Glu transporter control in both cell
lines even though strongly modulated by the upregulated Gln transporter
activity in the
LLC-PK1-F+
cells and, moreover, that because of this the
LLC-PK1 cell line is under tighter
Glu transporter control, at least with the 1.8 mM
L-Gln DMEM.

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Fig. 6.
Intracellular [14C]Gln
conversion to [14C]Glu
measured over 60 s in monolayers incubated with or without 5 mM
D-Asp for 45 min. Results are
means ± SE from 7 and 5 pairs of
LLC-PK1-F+
and LLC-PK1 plates, respectively.
* P < 0.05 vs. LLC-PK1-F+ and
** P < 0.05 vs. respective control.
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The rates for Gln uptake and NH+4 and Ala
formation measured over the 45-min time course are presented in Fig.
7. Steady-state Gln uptake was 2.2-fold
higher in the
LLC-PK1-F+
cell line (17.6 ± 0.8 vs. 8.1 ± 0.5 nmol · min
1 · mg
1,
P < 0.001), consistent with the
2.8-fold higher unidirectional uptake flux measured over 1 min (above).
Ala formation was 3.2-fold higher in the
LLC-PK1-F+
cell line (11.8 ± 1.2 vs. 3.7 ± 0.6 nmol · min
1 · mg
protein
1,
P < 0.01) in contrast to
NH+4 formation, which was only 1.6-fold
greater in the
LLC-PK1-F+
cell line (6.5 ± 0.5 vs. 4.1 ± 0.2 nmol · min
1 · mg
1,
P < 0.05). The large disparity
between Gln uptake and NH+4 formation in the
LLC-PK1-F+
cell line as opposed to Ala formation suggests that the Gln is largely
metabolized via the Gln transamination pathway under these conditions
(25). The effect of D-Asp on the
rates of NH+4 and Ala formation is
also shown in Fig. 7. In the
LLC-PK1-F+
cells, D-Asp increased
NH+4 formation 58% (10.3 ± 1.0 vs. 6.5 ± 0.5 nmol · min
1 · mg
1,
P < 0.001) and in the
LLC-PK1 cells 83% (7.5 ± 0.4 vs. 4.1 ± 0.2 nmol · min
1 · mg
1,
P < 0.001). The Ala formation rate
was unchanged in the
LLC-PK1-F+
cell line (11.8 ± 1.2 and 12.1 ± 1.3 nmol · min
1 · mg
1),
so that the ratio of NH+4 to Ala formation rose consistent with the intracellular Glu formed being metabolized predominantly through the GDH pathway and
NH+4 production rather than Ala formation. In
the LLC-PK1 cells, this effect is
more clear-cut due to the lower Ala formation apparently largely
coupled to the PDG flux and less to the Gln transamination pathway
activity (13). In these cells
D-Asp resulted in a small but
significant (P < 0.01) rise in Ala
formation (3.7 ± 0.6 to 4.5 ± 0.6 nmol · min
1 · mg
protein
1) consistent with
the increased Glu formation via PDG followed by transamination (above
Fig. 6); nevertheless, as in the
LLC-PK1-F+
monolayers, NH+4 formation increased even
further, so that the NH+4-to-Ala formation
ratio now rose from 1.1 to 1.7. Thus both cell lines respond to
D-Asp with an accelerated PDG
flux coupled predominantly to GDH and NH+4 formation.

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Fig. 7.
Gln uptake and NH+4 and Ala formation by
LLC-PK1-F+
and LLC-PK1 monolayers incubated
for 45 min in 1.8 mM L-Gln DMEM
or 1.8 mM L-Gln + 5 mM
D-Asp. Results are means ± SE from 15 pairs of
LLC-PK1-F+
control and D-Asp and 8 pairs of
similar LLC-PK1 plates.
* Difference between cell lines,
P < 0.05. ** Difference
between control and D-Asp,
P < 0.05.
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|
To test the Glu transporter regulation under conditions approximating
those found in vivo,
LLC-PK1-F+
monolayers were exposed to 0.9 mM
L-Gln, and the above
measurements were carried out. Interestingly, cellular Glu
concentration remained unchanged (Fig. 8)
compared with incubation in 1.8 mM
L-Gln (166 ± 13 vs. 175 nmol/mg protein); in contrast, cellular Gln content decreased nearly
50% compared with 1.8 mM L-Gln
(69 ± 12 vs. 140 ± 4 nmol/mg, respectively,
P < 0.001). Consequently, the ratio of Glu to Gln inside these cells doubled (2.4 vs. 1.2), consistent with
a greater degree of Glu transporter control over the glutaminase flux.
Indeed the estimated Gln-to-Glu conversion rate measured over 1 min was
markedly reduced at 0.9 mM (2.8 ± 0.3 vs. 6.5 ± 0.5 nmol · min
1 · mg
1,
P < 0.001). Note that now Gln uptake
(8.4 ± 1.0 vs. 17.6 ± 0.85 nmol · min
1 · mg
protein
1,
P < 0.01) and Ala formation (7.7 ± 0.2 vs. 11.8 ± 1.2 nmol · min
1 · mg
protein
1,
P < 0.01) were both reduced compared
with the respective rates at 1.8 mM
L-Gln. After 45 min in 5 mM
D-Asp, cellular Glu content had
fallen 28% (166 ± 13 to 120 ± 10 nmol/mg,
P < 0.01) without a change in the
Gln content, effectively lowering the Glu-to-Gln ratio (2.4 to 1.7).
The reduction in this ratio was associated with a 45% increase in the
glutaminase flux (2.9 ± 0.2 to 4.2 ± 0.3 nmol · min
1 · mg
protein
1,
P < 0.01), a 71% increase in
NH+4 formation (3.6 ± 0.5 to 6.1 ± 0.2 nmol · min
1 · mg
1,
P < 0.01), and a tendency for Ala
production to increase (7.7 ± 0.2 to 8.9 ± 0.7 nmol · min
1 · mg
protein
1,
P = 0.06), but nevertheless resulting
in a marked rise in the NH+4-to-Ala formation
ratio (0.47 to 0.69). Thus, under near-physiological medium Gln and Glu
concentrations, Glu transporter control of glutaminase flux appears
tighter as the Gln uptake rate falls off.

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Fig. 8.
Glu and Gln content of
LLC-PK1-F+
monolayers incubated in either 1.8 or 0.9 mM
L-Gln for 45 min. Results are
means ± SE.
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To assess the effect of upregulating extracellular Glu formation on PDG
and GDH fluxes in
LLC-PK1-F+
cells incubated in 0.9 mM L-Gln,
the
-GT-mediated conversion of extracellular Gln to Glu was enhanced
by PAH (1 mM). The effect of adding PAH to the medium was an elevation
in the monolayer Glu content measured after 45 min of incubation (189 ± 12 vs. 174 ± 18 nmol/mg for PAH and control, respectively,
P < 0.05) consistent with increased
extracellular formation and uptake. Although the medium Glu content
showed a net uptake (
0.37 ± 0.21 vs.
0.52 ± 0.18 nmol · min
1 · mg
1
for PAH and control, respectively), an increased rate of Glu formation
could in fact be demonstrated behind a
D-Asp block. In these
experiments monolayers were incubated with
D-Asp (5 mM) or
D-Asp plus PAH and the rate of
medium Glu accumulation was measured. In the presence of PAH, Glu
accumulation rate was 39% higher than in the
D-Asp-treated monolayers (3.40 ± 0.40 vs. 2.44 ± 0.26 nmol · min
1 · mg
1,
P < 0.02). The effect that
increasing the Glu uptake has on the steady-state formation of
NH+4 and Ala is presented in Fig.
9. In the presence of PAH overall
NH+4 formation increased 41% (4.1 ± 0.2 to 5.8 ± 0.4 nmol · min
1 · mg
1,
P < 0.01) without increasing flux
through the transamination pathway (5.2 ± 0.5 vs. 5.6 ± 0.6 nmol · min
1 · mg
1
for control and PAH, respectively). Note that the increased
NH+4 formation could not be accounted for by
the increased extracellular Gln breakdown (1.7 vs. 1.0 nmol · min
1 · mg
1
for increased overall NH+4 formation vs.
extracellular Gln hydrolysis). Consequently, the difference must be
derived from either the intracellular glutaminase flux or flux through the GDH pathway. However the estimated intracellular Gln breakdown rate
actually decreased 31% (2.2 ± 0.2 vs. 3.2 ± 0.2 nmol · min
1 · mg
1,
P < 0.05) consistent with the
elevated cellular Glu content and the shift of intracellular Glu into
the ammoniagenic pathway.

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Fig. 9.
NH+4 and Ala formation by
LLC-PK1-F+
monolayers incubated with 0.9 mM
L-Gln or 0.9 mM
L-Gln + 1 mM
p-aminohippurate (PAH) for 45 min.
Results are means ± SE from 6 pairs of monolayers.
* P < 0.05 vs. control.
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 |
DISCUSSION |
Our goal was to elucidate the role of the Glu transporter in modulating
cellular Gln metabolism, specifically its role in regulating flux
through the PDG and GDH pathways (Fig. 1, reactions 1 and 4). Our
previous studies using
LLC-PK1-F+
cells grown on porous supports had shown that either blocking or
slowing Glu entrance into these monolayers resulted in a 40-50% reduction in cellular Glu concentration and a two- to threefold increase in the intracellular conversion of Gln to Glu (32). In those
studies, Glu uptake was limited by using AT-125 to inhibit
-GT-generated extracellular Glu production or, alternatively, by
using D-Asp to inhibit Glu
uptake, both treatments for an 18-h period before assessing the PDG
flux. In the present design the parental
LLC-PK1 cell line was utilized as
a model for Glu availability-limited Glu transport, since
-GT
activity was only one-half that expressed in the Gln-adapted
LLC-PK1-F+
cell line (11, 12). Furthermore, these responses were studied after
only 45 min to assess whether acute regulation could, in fact, be
demonstrated. Consequently, on the basis of the available extracellular
Glu and the above hypothesis, we expected that the Gln flux through PDG
should be more suppressed in the Gln-dependent LLC-PK1-F+
cells, a paradoxical scenario given that these cells were selected to
grow on Gln as their sole energy source (12). Indeed, we too observed
the twofold higher
-GT activity as originally reported (11, 12) and,
as expected from the model shown in Fig. 1, an increase in cellular Glu
concentration in the Gln-dependent cell line (Fig. 4 and Ref. 32).
However, unexpected on the basis of Glu transport alone was the greater
flux through the PDG reaction in the Gln-adapted
LLC-PK1-F+
cells. How is this discrepancy to be explained? First, we had overlooked the obviously implied possibility that competitive inhibition requires factoring Gln uptake into the model, and second, if
the cellular L-Gln concentration
were higher in the
LLC-PK1-F+
cells as the result of Gln transporter activity, then the actual PDG
flux would depend on the existing ratio of Glu to Gln. In fact, the
ratio of inhibitor Glu to substrate Gln was approximately half that
found in the LLC-PK1 cell line,
and as a consequence flux through the PDG reaction was actually higher
in the
LLC-PK1-F+
cells in line with a greater dependence on Gln as a fuel source.
The benefit of framing the hypothesis shown in Fig. 1 and having two
closely related cell lines expressing very different
-GT activity is
that potentially more of the underlying control mechanism is revealed,
in this case the importance of the previously unrecognized adaptive
increase in L-Gln uptake. In the
original hypothesis (32), we had overlooked the adaptive nature of the role of Gln transporter(s) in maintaining cellular Gln simply because
our studies were limited to the
LLC-PK1-F+
cell line. In the present comparative study, the Gln-adapted cells
expressed a two- to threefold higher Gln uptake (Fig. 1, reactions 6 and
7), whether measured as
unidirectional Gln transport or as steady-state uptake (Fig. 7). Of
course, the two- to threefold increase in Gln transport would be
expected to elevate the cellular Gln concentration, overriding the
40-50% increase in cellular Glu, as in fact we observed (Figs. 4
and 5). That this was dependent primarily on Gln transport activity was
supported by the fall in cellular Gln with reduced transport activity
at the near-physiological medium Gln concentration. Previous studies
(24, 26) had shown that the
LLC-PK1 cell line expressed a
number of transport systems capable of transporting Gln, namely,
systems A, ASC, and L. The fact that the adaptive Gln uptake was
paralleled by similarly elevated Ala release (Fig. 1,
reaction 6) is consistent with
transstimulation of Gln uptake by intracellular Ala (5). Note that
although both
-GT and PDG pathways were enhanced in the
LLC-PK1-F+
cells and may yield Ala, their combined activity would not account for
the large Ala production, raising the question as to the actual pathway
metabolizing the Gln. A likely possibility is Gln transamination by the
so-called glutaminase II pathway, actually an initial transamination followed by deamidation (6, 25). According to these reactions (Fig. 1,
reaction 3), Gln would first undergo
transamination, forming Ala and
-ketoglutaramate, a
product that secondarily undergoes deamidation via an
-amidase. That
this pathway, at least the transamination step, is operative in the
parental LLC-PK1 cell line was
clearly shown by use of
15N-amino-labeled Gln; Sahai et
al. (25) found 15N-labeled Ala and
accumulation of
-ketoglutaramate, suggesting that the secondary
-amidase reaction is only loosely coupled to the transamination step
(7). If so, the elevated Gln uptake exhibited by the
LLC-PK1-F+
cells might be metabolized to Ala via this pathway with Ala efflux coupled to and driving the high Gln influx (Fig. 1,
reaction 6) (5). Note that, in the
LLC-PK1 cell line, this pathway
was not ammoniagenic, so that a considerable discrepancy exists between Ala and NH+4 formation consistent with what
we observed (Fig. 7). Thus an adaptive increase in Gln transport coupled to Gln transamination yielding Ala would readily account for
the enhanced Gln utilization observed to occur in the
LLC-PK1-F+
cell line.
A truer assessment of the Glu transport regulation of PDG flux required
experiments performed at the near-physiological Gln concentration (0.9 vs. ~0.8 mM for in vivo) (3). Under this condition, Glu transport
regulation of PDG flux is much tighter as both Gln uptake and the
cellular concentration declined ~50% (Fig. 8). In line with
suppressed glutaminase flux, both intracellular Glu formation and
NH+4 production were reduced ~50% compared
with the rates at 1.8 mM L-Gln.
Note that the fall in cellular Gln concentration was associated with a
decrease in Ala release, suggesting that Gln utilization by the Gln
transamination pathway is concentration dependent as previously noted
(6). Under these conditions, blocking Glu uptake with
D-Asp or enhancing uptake by PAH
activation of extracellular Glu production resulted in a corresponding
drop or elevation in cellular Glu. In response, Gln flux through PDG
either rose or fell, demonstrating regulatory control of this
rate-limiting reaction via the Glu transporter.
We also considered an additional role of the Glu transporter activity,
apart from the transfer of
L-Glu, that potentially accelerates Glu flux through the GDH pathway (Fig. 1,
reaction 4). The basis for this is
the Glu transporter's acidifying effect (15) and the enhancement of
flux through the GDH pathway with cellular acidosis (19, 21, 25). In
cells expressing competing GDH and alanine aminotransferase pathways
(Fig. 1, reactions 4 and
5), acidosis would favor flux
through GDH, so that the ratio of NH+4 to Ala
formation rises as a consequence (19). We thus deployed both the
NH+4 formation rate and this ratio as indexes
of GDH flux under these conditions in the two cell lines; in addition,
these indexes were monitored at 0.9 mM
L-Gln to reduce the high
"background" Ala formation in the
LLC-PK1-F+
cell line. To ensure a high transporter turnover, we used 5 mM D-Asp, which is readily
transported (1), or enhanced extracellular Glu formation to drive the
transporter. Under this condition (fall in cellular pH),
NH+4 production increased and to an extent
greater than that attributed to either the increased flux through the
PDG pathway, as is the case for
D-Asp, or to the increased
-GT-associated NH+4 formation, as is the
case for PAH. These findings are therefore consistent with increased
Glu deamination and NH+4 formation as a
consequence of Glu transporter activity and cellular acidosis. In
contrast, Ala formation remained unchanged or barely increased, indicative of a near-constant flux through the transamination pathway.
Consequently, in the LLC-PK1 cell
line, the NH+4-to-Ala formation ratio rose
from 1.1 to 1.7, whereas for
LLC-PK1-F+
cells, at 0.9 mM L-Gln, the
ratio rose from 0.5 to 0.7. Thus these findings are consistent with and
predicted from the Glu transporter's role in acidifying the cells and
accelerating the GDH ammoniagenic flux.
Finally, D-Asp clearly promoted
the efflux of L-Glu from the
cells as shown by the accumulation of medium Glu with both
-GT and
Glu uptake blocked. In this regard, it should be noted that the Glu
transporter expressed in plasma membrane vesicles exhibits an exchange
reaction of extracellular D-Asp
for L-Glu (8, 28); thus an
exchange reaction might account for the high Glu efflux and
accumulation in the medium. On the other hand, its also possible that
Glu efflux is proton driven (20), so that
D-Asp transport, by delivering
an acid load, might indirectly effect Glu efflux as well as flux
through GDH. Noteworthy, acidogenic hormones, i.e., growth and
parathyroid hormones (22, 31) as well as prostaglandin
E2 (unpublished observation), have
been shown to mimic these effects on cellular metabolism, suggesting
that this mechanism may have physiological and pathophysiological
relevance. Thus the Glu transporter may play yet another role in
cellular Glu metabolism by shunting metabolically generated Glu out
into the medium. Although we do not know the source of the large Glu efflux from these cells in the presence of
D-Asp, it is not unreasonable to
suspect that a major fraction is derived from the intracellular Gln
conversion to Glu; if so, that would place PDG-generated Glu in a
cytosolic pool accessible to the transporter as suggested by Kvamme et
al. (18) and possibly explain why plasma membrane transporters exert
this extraordinary control over a major metabolic pathway. Further
studies utilizing labeled Gln and cellular pH measurements will be
required to elucidate the source and mechanism underlying this
obviously important transporter-mediated Glu efflux.
The excellent secretarial and technical support of Dawn Powell and
Liesl Milford is gratefully acknowledged.
Acivicin (AT-125) was a generous gift from Dr. Michaele Christian,
Chief of the Investigational Drug Branch of the National Cancer
Institute, Division of Cancer Treatment, Diagnosis and Centers.
Address for reprint requests: T. C. Welbourne, Dept. of Physiology,
LSUMC-Shreveport, PO Box 33932, Shreveport, LA 71130-3932.