From the Division of Endocrinology and
¶ Division of Child Development and Pediatric Rehabilitation,
Children's Hospital of Philadelphia and § Department of
Biochemistry and Biophysics, School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, October 16, 2002
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ABSTRACT |
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Glutamate dehydrogenase (GDH) is regulated
by both positive (leucine and ADP) and negative (GTP and ATP)
allosteric factors. We hypothesized that the phosphate potential of
In addition to glucose, amino acids and other metabolic fuels are
important stimulants of insulin secretion from pancreatic GDH has also been proposed by Maechler and Wollheim (6) to play an
essential role in glucose-mediated insulin secretion by acting in the
reverse direction to catalyze production of glutamate, which is
hypothesized to work as a cofactor in the process leading to exocytosis
of insulin granules. These investigators have suggested that lower
levels of GDH in mouse islets may explain the reduced second phase of
insulin secretion of mouse islets compared with the predominant second
phase insulin release observed in rat and human islets (7). Their
suggestion that net flux through GDH is toward synthesis rather than
oxidation of glutamate has been contradicted by other reports (8-10)
and is also not consistent with our proposal that the mutations of GDH
that cause hyperinsulinism in children act by increasing the oxidation
of glutamate and, presumably, the resulting decrease of the
intracellular glutamate level. Because of the renewed interest
in GDH as a potential key regulatory step in amino acid- and
glucose-stimulated insulin secretion and because of the controversy
about the direction of net flux through the enzyme, the present
experiments were undertaken to test the hypothesis that LSIS is due to
increased oxidation of glutamate by GDH and that this process is
inhibited by glucose.
Islets Isolation and Culture--
Adult male Wistar rat islets
were isolated by collagenase digestion and cultured in RPMI 1640 medium
(glucose-free; Sigma). The culture medium was supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 units/ml
penicillin, and 50 µg/ml streptomycin, and islets were incubated at
37 °C in a 5% CO2/95% air humidified incubator. Islets
were cultured with different concentrations of glucose, 5, 10, and 25 mM for 3 to 4 days.
Insulin Secretion by Perifused Islets--
100 cultured rat
islets were loaded onto nylon filters in a small chamber and perifused
in a Krebs-Ringer bicarbonate buffer (115 mmol/liter NaCl, 24 mmol/liter NaHCO3, 5 mmol/liter KCl, 1 mmol/liter
MgCl2, 2.5 mmol/liter CaCl2, pH 7.4) with
0.25% bovine serum albumin at a flow rate of 2 ml/min. Perifusate
solutions were gassed with 95% O2/5% CO2 and
maintained at 37 °C. Samples were collected every minute for insulin
assay. Insulin was measured by radioimmunoassay.
Leucine Oxidation--
Islets were cultured with different
concentration of glucose for 3-4 days. Batches of 100 islets were
preincubated with glucose-free Krebs-Ringer bicarbonate buffer
containing 2 mM glutamine for 60 min. Islets were then
incubated with 2 mM glutamine and different concentrations
of leucine for another 60 min with 1 µCi of
[1-14C]leucine (PerkinElmer Life Sciences)
present. A trap filter was placed in each tightly sealed glass tube to
collect the 14CO2 produced by the islets, and
the amount of radioactivity was determined by liquid scintillation counting.
GDH Activity and ATP Assays--
Islets were cultured with 10 mM glucose for 3 days. Batches of 250 islets for each
condition were then incubated with glucose-free Krebs-Ringer
bicarbonate buffer or 25 mM glucose for different time
periods up to 120 min. Islets were then homogenized in 70 µl of
buffer (0.1 M Tris acetate buffer with 1% Triton X-100, pH
8.0). GDH activity and ADP responsiveness were determined using the
assay described by Wrzeszczynski and Colman (11) with modifications as
described previously (2). Separate batches of 100 islets were taken to
measure islet ATP. After brief centrifugation, 10 µl of 5%
trichloroacetic acid was added to the islet pellet and maintained at
room temperature for 5 min. The sample was then homogenized after
adding 90 µl of buffer (0.1 mM Tris acetate buffer with
1% Triton X-100, pH 8.0). Samples were stored at Studies with [15N]Glutamine--
Islets were
cultured with 10 mM glucose for 3 to 4 days. Batches of
1,000 islets were first preincubated with unlabeled 10 mM
glutamine in Krebs-Ringer bicarbonate buffer for 90 min at 37 °C.
The islets were then incubated under the following conditions for
another 120 min: 10 mM [2-15N]glutamine
(Cambridge Isotope Laboratories, Inc., Andover, MA) as control.
For the other conditions the medium contained, in addition to 10 mM [2-15N]glutamine, the following
components: 10 mM leucine, 25 mM glucose, and
10 mM leucine and 25 mM glucose. Another group
of islets was sampled at time 0 with 10 mM
[2-15N]glutamine alone, the tube being placed on ice
immediately after adding the buffer containing 10 mM
[2-15N]glutamine. After incubation for 120 min, medium
was sampled for total ammonia, insulin, and
[15N]ammonia enrichment assays. Pellets were
suspended and homogenized with 200 µl of 6% perchloric acid,
neutralized to pH 7.0 with 1 M
K2CO3 and centrifuged to remove the potassium
perchlorate precipitate. The supernatant was used for determination of
amino acids, as well as 15N enrichments.
Assays for Amino Acids and Ammonia--
Ammonia was determined
by a sensitive diffusion method on Vitros Chemistry Analyzer using
Vitros AMON slide (Ortho-Clinical Diagnosis, Rochester, NY), which has
a sensitivity of <2 µM. Intracellular amino acids were
determined by high pressure liquid chromatography.
15N Analysis--
Gas chromatography-mass
spectrometry measurements of 15N isotopic enrichment (atom
% excess, APE) were performed as described previously (12) using the
Hewlett-Packard 5990 gas chromatography-mass spectrometry system.
15NH3 was determined following conversion of
ammonia to norvaline. 15N enrichment in amino acids was
determined using the t-butyldimethyl-silyl derivatives.
Materials--
All chemicals were from Sigma except where
indicated otherwise.
Calculations of Stable Isotope Studies--
The accumulation of
15N-labeled metabolites is obtained by the product of their
isotopic enrichment (atom % excess, APE, %) and their tissue
concentrations (nmol/1000 islets). Ammonia production (P-NH4+) was calculated according to the
equation, P-NH Data Analysis--
All the data are presented as mean ± S.E. Student's t tests were performed when two groups were
compared. One way analysis of variances was used when multiple groups
were compared. Differences were considered significant for
p < 0.05. To determine the threshold concentration of
stimulated insulin secretion in ramp perifusion studies, a t
test was used comparing the rates of each time point with base-line
insulin secretion rates. The first point that was significantly
different from base line was considered the threshold.
Effects of Glucose on LSIS--
Fig.
1 shows the insulin secretory responses
of cultured rat islets to leucine, KIC, or glucose after perifusion in
glucose-free buffer to allow run-down of energy stores for either a
short (50-min) or a long (120-min) period. Islets cultured with medium
containing 10 mM glucose followed by a short period of
glucose depletion (50-min) failed to respond to 10 mM
leucine (Fig. 1A). In contrast, islets exposed to the longer
period of glucose depletion (120-min) had a brisk, biphasic secretion
of insulin in response to 10 mM leucine. After the longer
periods of glucose depletion, base-line insulin release was 0.8 ± 0.1 ng/100 islets/min, and following leucine, the insulin secretion
reached a peak of 6.8 ± 1.6 ng/100 islets/min followed by a
second phase plateau of 10 ± 0.1 ng/100 islets/min.
In contrast to leucine, both 10 mM KIC and 10 mM glucose were able to stimulate biphasic insulin release
in islets exposed to glucose-free medium for 50 min (Fig. 1,
B and C). Glucose stimulated nearly identical
insulin responses after the short or long periods of energy depletion
(Fig. 1B). KIC at 10 mM caused a greater initial insulin peak (21 ± 4 versus 10 ± 4 ng/100
islets/min, p < 0.001) and a higher second phase
plateau (11 ± 0.3 versus 6 ± 0.2 ng/100 islets/min, p < 0.001) in islets with 120-min run-down
compared with islets with 50-min run-down (Fig. 1C).
Fig. 2 shows the effect of short or long
periods of glucose depletion on islet sensitivity to stimulation with a
leucine ramp. The threshold for stimulation of insulin release by
leucine was lower in islets with 120-min run-down than islets with
50-min run-down (6 versus 14 mM leucine).
Furthermore, maximum leucine-stimulated insulin secretion was doubled
by extended glucose depletion (15 ± 1 versus 8 ± 3 ng/100 islets/min, p < 0.001).
As shown in Fig. 3, the suppression of
leucine-stimulated insulin release by glucose was
concentration-dependent. When glucose was added to the
perifusion buffer during the 120-min equilibration period and then
removed for 20 min prior to stimulation with leucine, insulin release
was partially inhibited by 10 mM glucose and completely suppressed by 25 mM glucose. In contrast, exposure to
glucose had no effect on insulin release in response to depolarization with potassium chloride. When the glucose concentration of the medium used to culture isolated rat islets for 3 days prior to perifusion was reduced from 10 to 5 mM, a biphasic insulin
response to stimulation by 10 mM leucine was observed even
after very brief glucose depletion (data not shown).
Fig. 4 shows the effect of increasing
glutamine concentrations on glucose suppression of leucine-mediated
insulin release. As shown in panel A, glucose suppression of
LSIS was overcome in a dose-dependent manner by increasing
the concentration of glutamine from 0 to 10 mM. In the
absence of leucine, glutamine alone, even at 10 mM, did not
stimulate insulin release. As shown in panel B, increasing
the concentration of leucine from 10 to 20 mM increased
insulin secretion in the presence of 5 and 10 mM glutamine
but had very little added effect in the presence of 2 mM
glutamine. These results showed that high leucine in the presence of
high glutamine could overcome glucose inhibition of LSIS.
To test whether the inhibitory effect of glucose on LSIS was on the
pathway of leucine oxidation via KIC, the effect of glucose pretreatment on oxidation of [1-14C]leucine in the
absence of glucose was examined (Fig. 5).
Maximum rates of leucine oxidation were lowered by 30% with increasing concentrations of glucose pretreatment (p < 0.01).
However, oxidation rates of 20 mM leucine by islets
cultured in 10 mM glucose were 80% the rate with 10 mM leucine in islets cultured at 5 mM glucose. Studies of insulin secretion showed that islets cultured in 10 mM glucose did not respond to 20 mM leucine,
whereas islets cultured with 5 mM glucose responded to 10 mM leucine. Thus, it seems that direct oxidation of leucine
contributes little or nothing to the process responsible for LSIS.
Effects of Glucose Depletion on Islet ATP and GDH--
To
determine the factors responsible for altering leucine sensitivity, we
measured the effects of incubation in glucose-free medium for
varying time on islet ATP concentrations and on basal and
maximal GDH activity. As shown in Table
I, withdrawal of glucose for 120 min
resulted in a nearly 50% fall in islet ATP concentration. Basal GDH
enzyme activity was ~20% of the ADP-stimulated maximal value,
suggesting carryover of inhibitors, such as GTP, in the crude islet
homogenate. Basal GDH activity increased during the 120 min of glucose
depletion by 40% without a change in maximal enzyme activity,
consistent with a decline in islet content of GTP and ATP, which are
potent allosteric inhibitors of the enzyme.
Effects of Leucine and Glucose on Flux through GDH--
The above
experiments identified GDH as the likely site for glucose suppression
of leucine-mediated insulin release. To examine in detail the effects
of leucine and glucose on rates of flux through GDH, experiments were
carried out using 2-15N-labeled glutamine to trace the flow
of the amino nitrogen into glutamate and ammonia. A 90-min
pre-incubation in unlabeled 10 mM glutamine was used to
mimic the condition of prolonged glucose depletion. Incubations with 10 mM [2-15N]glutamine alone or together with
leucine, glucose, or leucine plus glucose were then carried out for
2 h using batches of 1,000 isolated rat islets per tube. As shown
in Table II, both leucine and glucose
stimulated insulin secretion by these islets. However, the combination
of the two gave the same rates of insulin release as glucose alone,
consistent with glucose inhibition of LSIS seen previously. The
intracellular concentrations of glutamine, glutamate, aspartate,
alanine, and
As shown in Table III, about 60% of
islet glutamate and aspartate were replaced from
[15N]glutamine in the control and glucose
incubations. The isotopic enrichment of glutamate and aspartate was
decreased by 10-20% in the presence of leucine, consistent with some
contribution of unlabeled nitrogen from leucine through transamination.
Glucose decreased the concentrations but not the isotopic enrichment of both glutamate and aspartate, indicting that glucose suppressed flux
from glutamine through glutaminase into these amino acids. Isotopic
enrichment of GABA was not determined, but turnover of this pool was
likely to have been small, because the concentrations of GABA remained
essentially unchanged under all of the incubation conditions (Table
II).
As shown in Table III, total ammonia production from the combination of
the glutaminase and GDH reactions was stimulated by leucine and
suppressed by glucose. In the presence of glucose, the stimulation of
ammonia release by leucine was inhibited. The isotopic enrichment of
ammonia was significantly increased by incubation with leucine,
confirming increased flux through both the glutaminase and glutamate
dehydrogenase steps.
Table III shows the calculated rates of 15N-labeled
glutamate and aspartate production from glutamine and of the flux rates
through the glutaminase and GDH steps. Production of glutamate and,
especially, aspartate were decreased by incubation with leucine
compared with control and further suppressed by incubation with glucose
or glucose plus leucine. Leucine stimulation produced a 350% increase
in flux through GDH and a 140% increase in flux through glutaminase compared with the control islets. In contrast, glucose stimulation of
islets was associated with a 50% reduction in flux through both GDH
and glutaminase. The stimulatory effects of leucine on GDH and
glutaminase flux were blocked in the presence of glucose, consistent
with the observation that glucose also blocked leucine-stimulated insulin release.
The discovery of the GDH linked form of hyperinsulinism has made
it important to explore the mechanisms of increased protein and leucine
sensitivity of insulin secretion observed in patients with this
disorder. The present report describes a robust experimental model in
isolated cultured rat islets using a paradigm of energy depletion, or
run-down, to test the sensitivity for LSIS. The results show that the
energy potential regulates glutaminolysis and modifies the sensitivity
of -cells regulates the sensitivity of leucine stimulation. These
predictions were tested by measuring leucine-stimulated insulin
secretion in perifused rat islets following glucose depletion
and by tracing the nitrogen flux of
[2-15N]glutamine using stable isotope techniques.
The sensitivity of leucine stimulation was enhanced by long time
(120-min) energy depletion and inhibited by glucose pretreatment. After
limited 50-min glucose depletion, leucine, not
-ketoisocaproate,
failed to stimulate insulin release.
-Cells sensitivity to leucine
is therefore proposed to be a function of GDH activation. Leucine increased the flux through GDH 3-fold compared with controls while causing insulin release. High glucose inhibited flux through both glutaminase and GDH, and leucine was unable to override this
inhibition. These results clearly show that leucine induced the
secretion of insulin by augmenting glutaminolysis through activating
glutaminase and GDH. Glucose regulates
-cell sensitivity to leucine
by elevating the ratio of ATP and GTP to ADP and Pi and
thereby decreasing the flux through GDH and glutaminase. These
mechanisms provide an explanation for hypoglycemia caused by mutations
of GDH in children.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells.
Leucine, which has been studied intensively, may stimulate insulin
release through two different mechanisms. The first involves transamination of leucine to
-ketoisocaproate
(KIC)1 and subsequent
mitochondrial oxidation. The second promotes insulin release via
allosteric activation of glutamate dehydrogenase (GDH) causing
oxidation of glutamate to the Krebs cycle intermediate,
-ketoglutarate, plus ammonia. The importance of the latter
mechanism has been highlighted recently by the discovery of a dominant
form of congenital hyperinsulinism associated with mutations of GDH leading to a gain of enzyme activity, because sensitivity to inhibition by GTP and ATP is impaired (1-3). Affected children have increased
-cell responsiveness to leucine and are susceptible to acute hypoglycemia following a high protein meal (4). The involvement of GDH
may explain the observation that, in contrast to other amino acids,
leucine-stimulated insulin secretion (LSIS) is suppressed by high
glucose. For example, Gao et al. (5) reported that glucose
inhibits leucine stimulation of glutaminolysis and insulin secretion in
isolated mouse islets, presumably by increasing intracellular ATP and
GTP while decreasing ADP and thus inhibiting GDH activity.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until
assay. ATP was assayed in triplicate by a luminimetric method using an
ATP assay kit (Enliten ATP assay kits; Promega).
blank-NH
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Effect of run-down duration on islet
responsiveness. Isolated rat islets were cultured with 10 mM glucose for 3 days and then perifused with 2 mM glutamine in the absence of glucose for run-down periods
of 50 min (diamonds) or 120 min (circles) prior
to stimulation with 10 mM leucine (panel A), 10 mM glucose (panel B), and 10 mM KIC
(panel C). Values represent the means ± S.E. for 100 islets from three separate perifusions.
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Fig. 2.
Effect of run-down duration on islet response
to leucine ramp. Isolated rat islets were cultured with 10 mM glucose for 3 days and then perifused with 2 mM glutamine in the absence of glucose for run-down periods
of 50 min (triangles) or 120 min (circles) prior
to stimulation with a leucine ramp (0 to 25 mM at 0.5 mM/min). Results are presented as means ± S.E. for
100 islets from three separate perifusions.
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Fig. 3.
Effect of glucose concentration on islet
responsiveness to leucine. Isolated rat islets were cultured with
10 mM glucose for 3 days and then perifused with 2 mM glutamine in the presence of different concentration of
glucose for 120 min prior to stimulation with 10 mM
leucine. Circles, 0 mM glucose;
diamonds, 10 mM glucose; triangles,
25 mM glucose. At the end of the experiment, after removal
of leucine for 10 min (glucose, 0 mM), islets were
stimulated with 30 mM KCl.
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Fig. 4.
Effect of leucine and glutamine concentration
on islet responsiveness. Isolated rat islets were cultured with 10 mM glucose for 3 days and then perifused in absence of
glucose for 20 min prior to expose to different concentration of
glutamine for 30 min prior to stimulation with 10 mM
(panel A) or 20 mM leucine (panel B).
Solid circles, 0 mM glutamine; solid
triangles, 2 mM glutamine; open triangles,
5 mM glutamine; open circles, 10 mM
glutamine.
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Fig. 5.
Effect of glucose on islet
[1-14C]leucine oxidation. Isolated rat islets were
cultured with 5, 10, or 25 mM glucose for 3 to 4 days.
Batches of 100 islets were preincubated with 2 mM glutamine
in the absence of glucose for 60 min and then incubated with
[1-14C]leucine for another 60 min. Solid
squares, 5 mM glucose; stars, 10 mM glucose; open circles, 25 mM
glucose. Values represent means ± S.E. from three to four
separate experiments.
Effect of run-down duration on islet ATP and GDH activity
-aminobutyric acid (GABA) were measurable under these
conditions. Islet glutamine, glutamate, aspartate, and alanine
concentrations remained relatively constant during the 2-h control
incubation with 10 mM glutamine. Incubation with 10 mM leucine did not change islet glutamate concentrations
but caused a 30% decrease in aspartate concentrations compared with control. Incubation with 25 mM glucose caused a 40%
decrease of glutamate and a 70% decrease of aspartate concentrations.
The combination of leucine plus glucose further decreased aspartate concentrations and partly reversed the glucose-induced depression of
glutamate concentrations. The intracellular alanine concentrations were
very small compared with the glutamate and aspartate pools. Alanine
concentrations rose in the presence of glucose, consistent with
increased glycolytic flux to pyruvate and subsequent transamination to
yield alanine.
Insulin secretion and islet amino acid concentrations
(nmol/1000 islets)
Rates of production of ammonia, [15N]glutamate, and
[15N]aspartate; 15N isotopic enrichments and flux
through GDH and glutaminase in cultured rat islets
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells to leucine stimulation. A prolonged period of run-down to
produce a state of energy depletion sensitizes the islet to leucine
stimulation. In contrast, with a short period of run-down after
withdrawal of high glucose, islets maintain a high energy potential and
are insensitive to leucine stimulation. The present results show that
the mechanism of LSIS is not primarily through the leucine oxidation
pathway, but, as illustrated in Fig. 6,
is because of increased glutamine catabolism with enhanced flux through
the glutaminase and GDH reactions. Glucose suppression of LSIS involves
inhibition of both of these two enzyme steps in glutamine oxidation by
an indirect mechanism involving changes in the concentrations of high
energy phosphates, GTP and ATP, and of ADP and Pi in
-cells.
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Fig. 6.
Effects of glucose on the pathways of
glutaminolysis and leucine-stimulated insulin secretion in
pancreatic -cells. As shown in
A, in the absence of glucose, the islet phosphate potential
decreases, leading to de-inhibition of both PDG and GDH, as
shown by the dashed line. In this state, GDH is responsive
to allosteric stimulation by leucine, as shown by the solid
line. Flux is increased from glutamine to glutamate and into the
Krebs cycle intermediate, a-ketoglutarate. When the supply of
acetyl-CoA is limited, the Krebs cycle stalls at oxaloacetate, leading,
by transamination with glutamate, to an accumulation of aspartate. In
contrast, as shown in B, in the presence of glucose,
elevation of the phosphate potential leads to inhibition of both PDG
and GDH and, as shown by the dashed line, renders GDH
insensitive to stimulation by leucine. The pathway of glutaminolysis
from glutamine to glutamate and to
-ketoglutarate is suppressed. The
end products of glycolysis, pyruvate, and acetyl-CoA sustain a complete
Krebs cycle, shifting the flow of aspartate toward glutamate formation
by transamination.
The results of the present experiments demonstrate that LSIS is
conditional, in contrast to the commonly held concept that leucine is
comparable with glucose in potency as an insulin secretogogue. These
studies clarify previous observations in the isolated perfused rat
pancreas showing that LSIS is induced by fuel depletion (13). The
conditional nature of LSIS is also consistent with observations in
humans showing that normal healthy subjects do not become hypoglycemic in response to intravenous leucine but become sensitive to leucine after treatment with a sulfonylurea drug, such as tolbutamide (14).
Because LSIS is mediated by allosteric activation of GDH (15), islet
responsiveness to leucine is directly related to the state of GDH
enzymatic activity. Thus, patients with the GDH-linked form of
hyperinsulinism are hypersensitive to leucine stimulation (1-3),
because of mutations that impair responsiveness to GTP and result in a
loss of negative allosteric regulation. In isolated mouse islets, Gao
et al. (5) have reported that leucine-induced elevation of
cytosolic calcium is blocked after treatment with high glucose. In
addition, in mouse islets, the enhancement of [U-14C]glutamine oxidation by the leucine analog
2-amino-2-norbornane-carboxylic acid, was inhibited by glucose
treatment (5). In the present study, the run-down paradigm clearly
showed that the dynamic changes in sensitivity of LSIS reflect the
regulation of GDH enzymatic activity by the -cells energy potential.
Fig. 6 illustrates some of the interactions revealed by the present
experiments in the glucose and leucine regulation of glutaminolysis and
flux through GDH. During post-prandial hyperglycemia, glucose is the
predominant energy source of -cells, and glucose metabolism increases the levels of ATP and GTP while decreasing the concentrations of ADP, GDP, and Pi (16, 17). The half-maximal inhibitory concentrations of GTP and ATP for allosteric inhibition of GDH are
50-100 nM and 10-20 µM, respectively (3).
These values are well below the intramitochondrial concentrations of
these nucleotides, implying that GDH activity might be totally
inhibited by the enhanced glucose metabolism following a meal. Under
such conditions of high energy potential, GDH becomes refractory to activation by leucine. During energy depletion, the ratio of ATP and
GTP to ADP and Pi decreases, and the sensitivity of GDH to allosteric stimulation becomes augmented. The data shown in Table I
suggest that the phosphate potential gradually decreases during islet
run-down until it reaches a critical threshold, at which point islets
become sensitive to leucine.
Previous studies of glutaminolysis in islets that have used [14C]glutamine as a tracer to follow the flux of glutamine into CO2 were unable to distinguish between transamination of glutamate and oxidative deamination. In the present study, by using [2-15N]glutamine we were able to directly follow the fate of the amino nitrogen of glutamine and the changes associated with leucine and glucose stimulation. The results highlight the fact that both glutaminase and GDH are regulated by the phosphate potential and are involved in the suppression of LSIS by glucose. Glutaminase, the pathway-controlling step in glutaminolysis, is a phosphate-dependent enzyme in islets (18). Thus, glucose may inhibit glutaminase by decreasing the inorganic phosphate level. Because glutamate is a strong inhibitor of glutaminase, leucine may indirectly stimulate the enzyme by removing glutamate as a result of activation of GDH (see Table III). Thus, regulation of glutaminolysis is the result of the inhibition or activation of these two enzymes.
Although the present experiments were designed to investigate ammonia
release from GDH deamination and the glutaminase reactions, some of the
alterations in islet amino acid metabolism observed during incubation
with glucose and leucine involve transamination reactions. For example,
under conditions in which islets were incubated with glutamine as the
sole fuel, the 15N tracer studies indicate that glutamine
is oxidized through glutamate, enters the Krebs cycle at
-ketoglutarate, and exits at oxaloacetate (OAA) by
transamination to aspartate through aspartate aminotransferase (AST)
(Fig. 6). This partial Krebs cycle pathway has also been demonstrated in brain (19). In this study, glutamate carbon accumulated
in the form of aspartate when glutamate was the sole carbon source, and
the transfer of carbons from glutamate to aspartate could be blocked by
the aminotransferase inhibitor, amino oxyacetate. In the present
experiments, addition of glucose or glucose plus leucine decreased the
ratio of aspartate to glutamate in islets, because the generation of
acetyl-CoA from glycolysis allows the Krebs cycle to proceed past
oxaloacetate, thus reducing the ratio of oxaloacetate to
-ketoglutarate and shifting the aspartate aminotransferase reaction
toward glutamate formation. The reduction of 15N labeling
of the glutamate and aspartate pools by addition of leucine probably
reflects donation of the leucine amino group by transamination to
glutamate and KIC. The fact that glucose inhibits LSIS but has little
effect on KIC-stimulated insulin release indicates that allosteric
activation of GDH is the primary mechanism by which leucine produces
insulin release, under the conditions of the present experiments.
The present experiments were designed to measure the oxidative deamination of glutamate through GDH. The results are compatible with the concept that the predominant direction of the GDH reaction in intact islets incubated with glutamine plus or minus leucine or glucose is toward glutamate oxidation. The present observations are consistent with studies by Cooper and co-workers (20, 21) in liver and by Yudkoff et al. (22) in brain indicating that the GDH reaction runs exclusively in the oxidative direction under normal conditions. As these investigators have pointed out, flux toward glutamate synthesis is not likely, because normal concentrations of ammonia are over 100 times lower than the Km for ammonia of the GDH reaction. The present experiments do not lend support to the hypothesis of Maechler and Wollheim (6) that the GDH reaction runs toward glutamate formation during glucose-stimulated insulin secretion. Indeed, under the conditions used, addition of glucose suppressed both glutamate concentrations and the activity of the GDH reaction. However, the present experiments cannot completely exclude the hypothesis put forth by Maechler and Wollheim (6), because direct measurements were not made of reductive amination flux through GDH.
The present experiments highlight the importance of a GDH-linked
metabolic network in -cells as illustrated in Fig. 6. The key
enzymes in this network include phosphate-dependent
glutaminase (PDG), GDH, and aspartate aminotransferase. An increased
phosphate potential leads to inhibition of both PDG and GDH, indicating that PDG and GDH may serve as intracellular energy sensors to regulate
amino acid metabolism. The metabolism of glucose may control the
intracellular amino acid homeostasis by changing the phosphate
potential. The changing of aspartate indicates that the transamination
reaction plays an important role in glucose and amino acid metabolism
in islets.
In conclusion, GDH and glutaminase play important roles in insulin
secretion stimulated by a mixture of glutamine and leucine. The
sensitivity of islets to leucine stimulation is tightly regulated by
the energy potential. GDH and glutaminase may serve as intracellular energy sensors to control the islet responsiveness to leucine stimulation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Henry Drott for technical assistance.
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FOOTNOTES |
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* These studies were supported in part by NIDDK, National Institutes of Health Research Grants RO1 DK 53012 and RO1 DK 56268 (to C. A. S.) and 22122 (F. M. M.), and by National Institutes of Health Grants HD 26979, and NS 37915 (to M. Y.). The work was presented in part at the 2001 and 2002 annual meetings of the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addresses: Division of
Endocrinology, Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-3421; Fax: 215-590-1605; E-mail: stanleyc@email.chop.edu.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M210577200
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ABBREVIATIONS |
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The abbreviations used are:
KIC, -ketoisocaproate;
GDH, glutamate dehydrogenase;
LSIS, leucine-stimulated insulin secretion;
APE, atom % excess;
GABA,
-aminobutyric acid;
PDG, phosphate-dependent
glutaminase.
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