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(Received for publication, May 15, 1995) From the
Previous studies indicated that in pancreatic islets the amount
of glucose-derived pyruvate that enters mitochondrial metabolism via
carboxylation is approximately equal to that entering via
decarboxylation and that both carboxylation and decarboxylation are
correlated with capacitation of glucose metabolism and insulin release.
The relatively high rate of carboxylation is consistent with the
current study's finding that pyruvate carboxylase is as abundant
in pancreatic islets as it is in liver and kidney. Since islets do not
contain phosphoenolpyruvate carboxykinase and, therefore, cannot carry
out glyconeogenesis from pyruvate, the carboxylase might be present in
the islet to participate in novel anaplerotic reactions. This idea was
first explored by incubating mitochondria from various tissues with
pyruvate. Mitochondria from tissues, such as pancreatic islets, liver,
and kidney, in which pyruvate carboxylase is abundant, exported a large
amount of malate and little or no citrate, isocitrate, and aspartate to
the medium. The amount of malate within the mitochondria was <1%
that in the medium. When pancreatic islet mitochondria were incubated
with [1-
Glucose is the most potent physiological insulin secretagogue
and metabolism of glucose via mitochondrial pathways is an essential
component of glucose's insulinotropic action. Recent work
indicated that the amount of glucose-derived pyruvate that enters
mitochondrial pathways via carboxylation is approximately equal to that
entering the citric acid cycle via decarboxylation in pancreatic
islets(1, 2) . Furthermore, the data suggested that
pyruvate carboxylation occurs via the pyruvate carboxylase reaction in
the mitochondrion rather than via the reverse of the malic enzyme
reaction in the cytosol(1, 3) . Pyruvate carboxylase
enzyme activity has long been known to be present in the pancreatic
islet(4) , and recent data(5) , including data herein,
indicate that the islet contains an amount of pyruvate carboxylase
equivalent to that in gluconeogenic tissues, such as liver and kidney.
Pyruvate carboxylase is not present in the islet for the purpose of
glyconeogenesis because the islet contains essentially no enzyme
activity (6, 7) or mRNA (8) for the
gluconeogenic enzyme phosphoenolpyruvate carboxykinase, which, along
with pyruvate carboxylase, catalyzes the conversion of pyruvate to
phosphoenolpyruvate. This information prompted me to look for a reason
for the abundance of pyruvate carboxylase in the islet. In the
current study, in order to view the mitochondrial effects of glucose
metabolism in isolation, mitochondria from pancreatic islets and other
tissues were incubated with pyruvate, the final cytosolic metabolite of
glucose which enters mitochondrial metabolism. Mitochondria from islets
and other tissues that contain pyruvate carboxylase exported malate to
the surrounding medium. When islet mitochondria were incubated with
[1- Pancreatic islets were isolated from fed Sprague-Dawley rats
that weighed 250-300 g as described
previously(1, 2, 3, 7, 8) .
Mitochondria were isolated from islets and other tissues by the method
of Johnson and Lardy (9) as described previously(10) .
Briefly, when mitochondria were isolated from islets, about 2,000
islets were homogenized in 0.4 ml of 70 mM mannitol, 230
mM sucrose, and 5 mM potassium Hepes buffer, pH 7.5
(MSH solution) and centrifuged at 600
Because commercially
available aspartate aminotransferase is contaminated with a minute
amount of alanine aminotransferase, which caused a high blank value in
the presence of high amounts of NAD(P) and/or glutamate or aspartate,
method B was developed. In addition, after the first several assays, a
beef liver mitochondrial aspartate aminotransferase preparation devoid
of alanine aminotransferase (a generous gift of L. A. Fahien) was used
when the method A assay was used.
Figure 1:
Relative amounts of pyruvate
carboxylase in tissues of the rat. Protein (29 µg/lane) from each
tissue was separated by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose, and probed with
Figure 2:
Export of malate and other metabolites
formed from pyruvate by mitochondria from various tissues. Mitochondria
from various tissues were incubated at 37 °C for 5 min, a zero time
sample was withdrawn and then pyruvate (5 mM) was added.
Subsequent samples were withdrawn at the intervals shown. Metabolites
within and outside mitochondria were estimated with standard
spectrophotometric assays as described under ``Experimental
Procedures.'' Results shown are from one of four similar
experiments.
Figure 5:
Export of
Figure 6:
Export of
Figure 4:
Export of
Figure 7:
Estimates of pyruvate carboxylation as
malate formation or
The large amount of
pyruvate carboxylase in islet mitochondria ( Fig. 1and Table 1) is even more convincing evidence for anaplerosis
occurring in the islet mitochondrion. Since the islet, unlike other
tissues in which pyruvate carboxylase is plentiful, such as liver,
kidney, and adipose tissue(11) , contains essentially no
phosphoenolpyruvate carboxykinase(6, 7, 8) ,
the companion enzyme to pyruvate carboxylase for the formation of
phosphoenolpyruvate from pyruvate, the islet cannot perform
glyconeogenesis. This suggests that pyruvate carboxylase in the islet
catalyzes an anaplerotic reaction in non-gluconeogenic pathways, which
are important for insulin secretion. It was, therefore, hypothesized
that carbon derived from pyruvate carboxylation must exit the
mitochondrion primarily as malate, since carbon in oxaloacetate,
malate, and fumarate probably equilibrates rapidly in islets (1) as in many other tissues, and malate is the only one of
these compounds actively transported across the inner mitochondrial
membrane(19) . Some carbon might also exit the mitochondria as
citrate and isocitrate. Once in the cytosol, malate could undergo
decarboxylation to pyruvate, which can then re-enter the mitochondrion
to form a cycle (Fig. 8). Such a pyruvate malate shuttle could
explain the apparent high estimates of carboxylation in pancreatic
islets obtained with the
Figure 8:
The pyruvate malate shuttle and pyruvate
decarboxylation.
Ashcroft (4) showed in 1970 that islets contain pyruvate
carboxylase enzyme activity, and the current study shows that the
amount and activity of the enzyme in the islet are equal to or slightly
exceed those in liver and kidney ( Fig. 1and Table 1),
which are tissues known for their abundance of this enzyme. We
previously obtained evidence that islets possess pyruvate carboxylase
mRNA (3, 8) and protein (5) , which are
up-regulated by glucose. The rate of carboxylation of glucose-derived
pyruvate in islets is proportional to the extracellular glucose
concentration (2) and thus insulin secretion. This and the
up-regulation of pyruvate carboxylase by glucose are consistent with
the idea that the enzyme is present in the beta cell of the islet. The idea for a pyruvate malate shuttle is based not only on the
pyruvate carboxylation studies in islets, but also on work from the
Lardy laboratory (22) in which it was demonstrated in 1966 that
when rat liver mitochondria are incubated with pyruvate and
Figure 3:
Export of malate and other metabolites
formed from pyruvate by pancreatic islet mitochondria. Mitochondria
from about 2,000 islets were washed once and incubated at 37 °C for
5 min, a zero time sample was obtained, pyruvate (5 mM) was
added, and samples were obtained at various intervals shown.
Metabolites in the medium outside the mitochondria were measured.
Conditions and methods were as described in the legend to Fig. 2except metabolites were measured fluorometrically. Results
shown are from one of three similar
experiments.
Although the
amounts of pyruvate-derived citrate and isocitrate exiting pancreatic
islet mitochondria were very small and at the level of detection in
this study, more of these compounds might be produced in cells
presented with a variety of nutrients in addition to glucose, such as
leucine or glutamine, as might occur in vivo. Citrate in the
cytosol could undergo a series of reactions widely believed to be an
important mechanism for NADPH formation required for fatty acid
synthesis(39, 40) . Citrate lyase could catalyze the
cleavage of citrate to oxaloacetate and acetyl-CoA. Oxaloacetate should
be reduced to malate via malate dehydrogenase thus forming NAD. Malate
could undergo oxidation to pyruvate producing NADPH, as described
above. This is in keeping with the known oxidized NAD/NADH ratio and
reduced NADP/NADPH ratio in the cytosol of most types of
cells(41) . Although this shuttle would appear to be minimally
active in beta cells metabolizing only glucose, it may be sufficiently
active to form acetyl-CoA needed to yield micromolar concentrations of
malonyl-CoA in the cytosol. The inhibition of carnitine palmitoyl-CoA
transferase by malonyl-CoA in the beta cell (42) and its
effects on fatty acid metabolism are part of an emerging and intriguing
story of metabolite regulation of insulin
secretion(43, 44, 45, 46, 47) . Isocitrate could produce NADPH and The
pyruvate malate shuttle should be far more effective than the pentose
phosphate pathway in producing NADPH in the cytosol. The pentose
phosphate pathway can produce NADPH only as rapidly as glucose enters
and completes its transit through the pathway. Unlike a metabolic
pathway, a shuttle is not regulated longitudinally and can replenish
its own reactants so it can operate at a very rapid pace. This idea is
in agreement with evidence from numerous studies, which indicates that
the pentose phosphate pathway is not very active in the pancreatic
islet. Less than 3% of glucose oxidation in the islet occurs via this
pathway(2, 48, 49, 50, 51) . The idea of malate, citrate, and isocitrate shuttles is relevant in
view of previous suggestions that redox state is important in
transducing metabolic stimuli into insulin secretion and implies that
reactions which utilize NADPH are important in the beta cell. Panten
and Ishida (52) reported in 1975 that NAD(P)H fluorescence
increases in pancreatic islets stimulated with glucose, and this has
been confirmed in islet cell preparations enriched with beta
cells(53, 54) . Ashcroft and Christie (21) proposed that the cytosolic NADPH/NADP ratio is increased
in glucose-stimulated islets, as judged from an increased
malate/pyruvate ratio, and Sener et al.(55) have
proposed that cytosolic NADPH formed in the malic enzyme reaction from
malate arising from mitochondrial metabolism of glutamate or leucine is
important for insulin secretion. The metabolism of glucose, however,
differs from the metabolism of the latter two compounds because
glucose-derived carbon cannot exit the citric acid cycle unless
anaplerosis occurs. Our data suggest that the pyruvate malate shuttle
is a major means of generating cytosolic NADPH from the metabolism of
glucose without depleting citric acid cycle intermediates. A number of
cytosolic enzymes that catalyze reactions in which NADPH is a cofactor
are known to be present in islets. These include glutathione reductase (56, 57) , ( The possible relevance of malic enzyme
and NADPH to beta cell function is accentuated by the work of
Coleman(68, 69) . He showed that the severity of the
diabetes produced by the mutation diabetes (db) in the mouse
is markedly strain-dependent. The insulin cells of strains possessing
an allele at the regulatory locus for malic enzyme that confers low
malic enzyme activity were unable to respond with sustained
hyperinsulinism to the insulin resistance caused by the extreme obesity
resulting from the db gene and suffered from severe diabetes
and islet atrophy. If malic enzyme levels are deficient in the beta
cell in these animals, inability to produce enough NADPH and other
metabolites could explain their severe diabetes.
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore by hereby
marked ``advertisement'' in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
Volume 270,
Number 34,
Issue of August 25, pp. 20051-20058, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
FURTHER IMPLICATION OF CYTOSOLIC NADPH IN INSULIN SECRETION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C]pyruvate, radioactive carbon appeared
in the medium primarily in malate. Very little radioactivity appeared
in amino acids, and little or no radioactivity appeared in citrate and
isocitrate. Carbon 1 of pyruvate can be incorporated into malate and
other citric acid cycle intermediates only via carboxylation, as this
carbon would be lost via decarboxylation when pyruvate enters the
citric acid cycle as acetyl-CoA via the pyruvate dehydrogenase
reaction. The amount of malate formed equaled the CO
formed and the radioactivity from C-1 of pyruvate recovered in
malate slightly exceeded the formation of CO in agreement with our previous studies that reported a high rate
of carboxylation of pyruvate in intact islets. When intact pancreatic
islets were incubated with methyl [U-C]succinate
as a mitochondrial source of four-carbon dicarboxylic acids,
radioactivity appeared in pyruvate and lactate. Taken together with
previous studies, the current results suggest that during
glucose-induced insulin secretion there is a shuttle operating across
the mitochondrial membrane in which glucose-derived pyruvate is taken
up by mitochondria and carboxylated to oxaloacetate by pyruvate
carboxylase. The oxaloacetate is converted to malate which exits the
mitochondrion, where, in the cytosol, it is decarboxylated to pyruvate
in the reaction catalyzed by malic enzyme. This pyruvate re-enters
mitochondrial pools. Such a cycle produces NADPH in the cytosol. Since
it is a cycle, this shuttle can produce far more NADPH than the pentose
phosphate pathway, which is known to be a very minor route of glucose
metabolism in the islet. If it is accepted that this shuttle is active
in the insulin cell, this implicates NADPH regeneration in insulin
secretion.
C]pyruvate, the rate of appearance of
radioactivity in malate and total malate formation roughly equaled the
formation of radioactive CO. Since incorporation of
radioactivity into malate could only occur via carboxylation, as C-1 of
pyruvate is lost when it undergoes decarboxylation, these results
support results of previous experiments in which a high rate of
pyruvate carboxylation was found in intact
islets(1, 2) . Intact islets also incorporated
radioactive carbon from four-carbon dicarboxylic acids into pyruvate
when the islets were incubated with a methyl ester of
[C]succinate as a source of mitochondrial
four-carbon dicarboxylic acids. In mitochondria from islets given
pyruvate, the production of citrate, isocitrate, and amino acids, or
incorporation of radioactivity from
[1-C]pyruvate into these compounds was
minuscule. These observations suggest that a pyruvate malate shuttle is
operative in the insulin cell. In this shuttle pyruvate enters the
mitochondrion and is converted to oxaloacetate by pyruvate carboxylase.
The oxaloacetate is converted to malate, which exits the mitochondrion
to the cytosol where it is decarboxylated to pyruvate in the reaction
catalyzed by malic enzyme to produce NADPH. Pyruvate then re-enters the
mitochondrion where it can participate in another turn of the shuttle
or undergo decarboxylation to acetyl-CoA and oxidation. This shuttle is
rarely mentioned in the literature, and its existence in any tissue has
only been inferred from indirect evidence.
g for 10 min.
The supernatant fraction was saved, and the resulting pellet was
re-homogenized in 0.4 ml of MSH solution and centrifuged. The islet
supernatant fractions were combined and centrifuged at 15,000
g for 10 min to obtain a mitochondrial pellet. When islet
mitochondria were to be incubated with pyruvate, the supernatant
fraction was centrifuged at 6,500
g for 10 min instead
of at 15,000
g for 10 min. Mitochondria from other
tissues were isolated from rats starved 24 h and were homogenized in
three volumes of MSH solution (9) and washed four times in MSH
solution by centrifuging at 15,000
g for 10 min before
they were suspended in incubation medium. The islet mitochondrial
pellet was washed once or not washed at all.
Pyruvate Carboxylase
The amount of pyruvate carboxylase
protein was estimated by probing nitrocellulose blots of
SDS-polyacrylamide gels with I-streptavidin as described
previously(5, 11) . Pyruvate carboxylase enzyme
activity was estimated in 50 µl of reaction mixture containing 10
µl of tissue homogenate, 2 mM Na
ATP, 10 mM MgCl, 100 mM KCl, 1 mM
dithiothreitol, 8 mM sodium pyruvate, 0.16 mM acetyl-CoA, 20 mM Na[C]HCO (0.1 mCi/mmol), 0.1% Triton X-100, and 100 mM Tris-HCl
buffer, pH 7.6, maintained at 37 °C(12) . The reaction was
started by adding tissue fraction and stopped after 30 min by adding 50
µl of 10% trichloroacetic acid. Eighty microliters of the resulting
mixture was transferred to a scintillation vial and allowed to
evaporate to dryness for 15 h to allow unreacted CO to evolve. Radioactivity incorporated into resulting carboxylic
acids was estimated by liquid scintillation spectrometry.Metabolites Exiting Mitochondria of Larger
Tissues
Mitochondria from organs of rats starved 24 h were
suspended in a volume of MSH solution roughly equal to the weight (v/w)
of the tissue from which they were obtained. The solution contained 2
mM NaADP, 5 mM KHCO, and 5
mM potassium phosphate, pH 7.3. The mixture was incubated at
37 °C with shaking, and 1 ml of medium was removed after 5 min
(zero time sample) and at intervals after pyruvate (5 mM as
sodium salt) was added and immediately centrifuged at 14,000 g for 1.5 min. The supernatant fraction was quickly removed
and treated with 0.1 ml of 0.92 M perchloric acid and
centrifuged again. The acid extract was then neutralized with about 0.1
ml of 0.92 M KOH and used for metabolite assays. Metabolites
were estimated by standard enzymatic spectrophotometric
assays(13, 14) .
Metabolites Exiting Islet Mitochondria
Malate,
citrate, and isocitrate produced by islet mitochondria were estimated
by alkali-enhanced fluorescence. Briefly, mitochondria from about 2,000
islets were incubated at 37 °C in 120 µl of a solution of 5
mM KHCO, 2 mM NaADP, and 5
mM potassium phosphate in MSH solution, pH 7.3. After 5 min,
30 µl of the incubation mixture was removed (zero time sample) and
pyruvate was added to the remaining incubation mixture to give a
concentration of 5 mM. Samples of incubation mixture (30
µl) were subsequently withdrawn at 15 and 30 min. Immediately after
samples were withdrawn, they were centrifuged at 14,000 g for 1.5 min. The supernatant fraction was quickly withdrawn and
treated with 10 µl of 0.92 M perchloric acid. This mixture
was centrifuged, and the supernatant fraction was neutralized to pH
7 with about 10 µl of 0.92 M KOH. The supernatant
fraction from the neutralized extract was used for fluorometric
metabolite assays(15) . To estimate malate, 30 µl of
extract was incubated for 20 min with 220 µl of 50 µM
NAD, 2 mM glutamate, and 2-amino-2-methyl propanol buffer, pH
9.9, 0.5 µl of malate dehydrogenase (5 mg/ml, stored in 50%
glycerol), and 0.5 µl of aspartate aminotransferase (2 mg/ml, that
was centrifuged to remove (NH
)SO and re-constituted in reaction mixture). Potassium phosphate
buffer, pH 11.9 (0.25 ml of a 200 mM solution), was added, and
the mixture was heated at 60 °C for 15 min to destroy NAD. Twenty
microliters of 1 M imidazole was added, and then 0.5 ml of 12 M NaOH containing 6 mM HO was
added. The mixture was heated at 60 °C for 15 min to develop
fluorescence. The test tubes were cooled to room temperature, and
fluorescence was estimated with a Farrand Ratio-2 fluorometer.
Fluorescence from blanks, which were extracts of medium without
mitochondria or of mitochondrial samples that were carried through the
assay without the addition of the enzymes, was subtracted from the
total fluorescence in order to estimate the formation of compounds
attributable to metabolism. Samples with various concentrations of NADH
and extracts with known amounts of malate produced by kidney
mitochondria were carried through the assay as standards. Citrate was
estimated similarly except that the reaction mixture contained 50
µM NADH, 40 µM ZnCl, 50 mM Tris-HCl buffer, pH 7.6, 0.5 µl of malate dehydrogenase, and
0.01 units of citrate lyase in a final volume of 250 µl. HCl (0.1 M) was added to destroy NADH, and HO was not present in the 12 M NaOH. Isocitrate was also
estimated similarly to malate except that the reaction mixture
contained 50 µM NADP, 100 µM
MnCl, 50 mM Tris-HCl buffer, pH 8.0, and 5 µg
of isocitrate dehydrogenase (from glycerol). NADP was destroyed, and
fluorescence of NADPH was enhanced as in the assay for malate.
Mitochondria from about 2,000 fresh pancreatic
islets were incubated in a microcentrifuge test tube in 50 µl to
170 µl of a solution of MSH containing 5 mM potassium
phosphate, 2 mM NaC Incorporation from
[1-C]Pyruvate into Metabolites Exiting Islet
MitochondriaADP, 5 mM
KHCO, and 2-5 mM
[1-C]pyruvate (10-28 mCi/mmol) at pH 7.3
and 37 °C as described in legends to individual figures. After
various intervals up to 30 min, mitochondria were separated from the
medium by centrifugation at 14,000 g for 1.5 min. The
supernatant fraction was removed, treated with 5 µl of 0.92 M perchloric acid, and centrifuged at 14,000
g for
2 min. The resulting supernatant fraction was removed and neutralized
with about 5 µl of 0.92 M KOH.
Paper Chromatography
Part of the extract (20
µl) was mixed with a solution of malate, pyruvate, lactate,
citrate, and isocitrate to give a concentration of 0.2 M of
each compound and applied to a corner of Whatman No. 1 paper (20 cm
20 cm) for two-dimensional chromatography in a solvent of 10
parts isobutyric acid and 6 parts 1 M NH
OH in each
dimension. The paper was allowed to dry, and compounds were identified
by UV light and (after metabolites were identified enzymatically) by
spraying with bromcresol green. Malate was identified by painting the
relevant area of the chromatogram with a solution of 0.2 mM NAD, 0.2 mM phenazine methosulfate, 0.3 mM nitro
blue tetrazolium, 10 mM glutamate, 0.15 mg/ml malate
dehydrogenase, 0.04 mg/ml aspartate aminotransferase, and 0.4 M 2-amino-2-methyl propanol buffer, pH 8.7. Lactate was identified
in the same manner with this solution, which contained no glutamate but
contained lactate dehydrogenase (0.05 mg/ml) instead of malate
dehydrogenase. The spots were cut out of the paper, and radioactivity
present in the spots was quantified by liquid scintillation
spectrometry.Enzymatic and Column Chromatography Method
A
Another estimate of C-1 of pyruvate incorporated into malate,
citrate, and isocitrate was obtained by converting carbon from malate
and citrate into aspartate and carbon from isocitrate into glutamate
and measuring the radioactivity in the two amino acids. A portion of a
neutralized extract (20-50 µl) was mixed with 2 ml of a
solution of 1 mM each of malate, citrate, and isocitrate and
applied to a 2-ml column of Dowex 50 8 (H
form) (100-200-mesh). This column was washed with 2 ml of
H
O, and the malate in one-fourth (1 ml) of the resulting
effluent was converted to aspartate by adding NAD and glutamate to the
column effluent to give concentrations of 2 and 40 mM,
respectively. The pH was adjusted to about 9.9 by adding
2-amino-2-methyl propanol buffer to give a 50 mM
concentration. Malate dehydrogenase and aspartate aminotransferase (0.1
mg/ml each) were added, and one-tenth of the reaction mixture was
diluted with water and placed in a cuvette in order to monitor the
progress of the reaction at 340 nm. After about 20 min at room
temperature, the reaction was complete and the volume of the reaction
mixture was doubled by adding water and applied to a second Dowex 50
8 (H
) column (1 ml). The column was washed
with 10 ml of H
O and aspartate adhering to the column was
eluted with 2 ml of 2 M KOH followed by 2 ml of
HO. The eluate was neutralized with perchloric acid.
Citrate and isocitrate washed through the first column were treated
similarly to malate. To convert citrate into aspartate a portion of the
first column sample was adjusted to pH 7.6 with Tris buffer and
incubated with 40 µM ZnCl, and 40 mM glutamate and citrate lyase (0.02 units) and aspartate
aminotransferase (0.1 mg/ml) for about 20 min and applied to a 1-ml
Dowex 50 8 (H
) column and eluted as described
above. To convert isocitrate into glutamate, a portion washed from the
first column was adjusted to about pH 7.5 with Tris buffer and then
MnCl
, NADP, and aspartate were added to give concentrations
of 0.1, 4, and 40 mM, respectively. Isocitrate dehydrogenase
(0.08 mg/ml) and 0.1 mg/ml aspartate aminotransferase were then added.
After about 20 min at room temperature, the mixture was diluted with
water and added to a 1-ml Dowex 50 8 (H
)
column and the column was treated as described above. Radioactivity was
also eluted from the first column with KOH, and this represented the
incorporation of
C into amino acids, such as aspartate.
Radioactivity in the neutralized eluates was estimated by liquid
scintillation spectrometry. In addition,
[C]malate was processed in companion columns
identically to the unknown samples to estimate recovery of malate.
Recovery was always 80% or more. Radioactivity in blanks, which were
medium alone, as well as extracts from medium exposed to mitochondria
that were carried through the analytical steps but without the addition
of enzymes, was subtracted from radioactivity in extracts carried
through the full procedure in order to estimate the incorporation of
radioactivity attributable to metabolism.Enzymatic and Column Chromatography Method B
Fifty
microliters out of 120 µl of the neutralized extract was added to
450 µl of a solution of 50 µM NAD, 2 mM glutamate, 1.5 mM cycloserine, and 50 mM 2-amino-2-methyl propanol buffer, pH 9.9. Cycloserine was present
to inhibit a small amount of alanine aminotransferase activity in the
aspartate aminotransferase preparation. The mixture was divided into
two 0.25-ml portions. To one portion, 2.5 µg of malate
dehydrogenase (from a glycerol stock solution) and 1 µg of
aspartate aminotransferase (reconstituted in the reaction mixture so as
to remove (NH)SO) was added. The
reaction was allowed to proceed 15-20 min at room temperature,
0.75 ml of HO was added to each portion, and the portions
were applied to a 1.5-ml Dowex 50 8 (H
form)
column (100-200-mesh). The columns were washed with 30 ml of
H
O and then aspartate was eluted with 1.5 ml of 2 M KOH followed by 1.5 ml of HO. The eluate was
neutralized with perchloric acid and the radioactivity present was
estimated by liquid scintillation spectrometry. Radioactivity in
samples without enzymes, as well as extracts from medium incubated
without mitochondria, which were processed identically to mitochondrial
samples, was subtracted from the radioactivity in samples from
incubations with mitochondria processed with enzyme, to give the
radioactivity attributable to [C]malate exported
by the islet mitochondria. The recovery of
[C]malate estimated from the recovery of
[C]malate (0.2 mCi) processed through companion
reaction mixtures and columns was always greater than 80%.
Radioactivity incorporated into citrate was estimated identically to
that for malate in a reaction mixture that contained 2 mM glutamate, 1.5 mM cycloserine, 40 µM ZnCl, and 50 mM Tris-chloride buffer, pH 7.6,
and, when added, 0.01 units of citrate lyase, and 1 µg of aspartate
aminotransferase. Incorporation of radioactivity into isocitrate was
also estimated similarly to that for malate in a reaction mixture that
contained 50 µM NADP, 100 µM
MnCl, 2 mM aspartate, 1 mM cycloserine,
50 mM Tris-HCl buffer, pH 8.1, 5 µg of isocitrate
dehydrogenase, and 1 µg of aspartate aminotransferase.[
About 2,000
islets were incubated in 150 µl of Krebs-Ringer bicarbonate buffer,
pH 7.3, containing 10 mM dimethyl
[U-C]Pyruvate and Lactate Formation
from Methyl [C]SuccinateC]succinate (equal parts of dimethyl
[1,4C]- and
[2,3C]succinate synthesized as described
previously (1) plus added unlabeled dimethylsuccinate) (final
specific radioactivity, 2 mCi/mmol) after 90 min at 37 °C, one half
of the incubation medium was quickly removed from above the islets and
brought to 1 ml with HO. The islets were quickly washed
three times in cold Krebs-Ringer solution and were treated with 40
µl of 0.92 M KOH, which was then neutralized with about 30
µl of 0.92 M perchloric acid. The mixture was centrifuged,
and the supernatant fraction was removed and brought to 1 ml with
HO. Each of the 1-ml samples was applied to a 1.5-ml Dowex
50 8 column (H
form) (100-200-mesh),
which was washed with 2 ml of H
O. Three milliliters of
effluent was collected, and the pH of the sample was adjusted to
9.5-9.9 with about 2 µl of 30% KOH. NAD, glutamate, and
2-amino-2-methyl propanol buffer, pH 9.9, were added to give
concentrations of 1, 5, and 20 mM, respectively. Both samples
were divided into three equal parts of 1 ml. One part received lactate
dehydrogenase (25 µg), one part received lactate dehydrogenase plus
alanine aminotransferase (10 µg), and one part received no enzymes.
After 20 min at room temperature, the pH of the samples was adjusted to
7 with HCl and they were added to 1-ml Dowex 50 columns. The
columns were washed with 12 ml of water, and alanine was eluted from
the columns with 1.5 ml of 2 M KOH followed by 1.5 ml of
H
O. The radioactivity in the sample without enzymes was
subtracted from those with enzymes to enable calculation of the
radioactivity incorporated into lactate (fraction with lactate
dehydrogenase) and lactate plus pyruvate (fraction with both enzymes).
[C]Lactate and
[C]pyruvate were processed through companion
columns to estimate the recovery of these compounds. Recovery was
always greater then 80%.Malate and CO
The
unwashed mitochondrial pellet from about 2,000 islets was incubated in
the presence of [1- Formation from
[1-C]Pyruvate by Islet MitochondriaC]pyruvate exactly as
described above, except that the microcentrifuge test tube containing
the incubation mixture was maintained in a rubber-stoppered
scintillation vial. After 30 min at 37 °C, 0.5 ml of tissue
solubilizer was added to the scintillation vial and 60 µl of 0.92 M perchloric acid was added to the incubation mixture in the
microcentrifuge test tube. CO absorbed into
the tissue solubilizer over 3 h was estimated by liquid scintillation
spectrometry as described for studies with whole
islets(1, 2, 3) . There was 95% recovery of CO from companion test tubes to which
Na[C]HCO was added. The acid extract
was removed from the protein pellet and neutralized with 30% KOH.
Malate in the extract was estimated by alkali-enhanced fluorescence,
and C incorporation into malate was estimated by method B
as described above.Protein
Protein was estimated by the method of
Lowry (16) after precipitation with trichloroacetic acid.Materials
NAD, NADH, NADP, and NaADP
were from P-L Laboratories, Milwaukee, WI. Malate dehydrogenase (in 50%
glycerol), aspartate aminotransferase, alanine aminotransferase, and
isocitrate dehydrogenase (all from pig heart), rabbit muscle lactate
dehydrogenase, and Aerobacteraerogenes citrate lyase
were from Boehringer Mannheim. [1-C]Pyruvate,
Na[C]HCO, and
[1,4(2,3)-C]malate were from Amersham.
[1,4-C]Succinate,
[2,3-C]succinate,
[2-C]pyruvate, and Solvable Tissue Solubilizer
(0.5 M) were from DuPont NEN. Sodium pyruvate was from Sigma.
Pure rat adipocyte pyruvate carboxylase was a gift of Dr. C. J.
Lynch(11) .
Pyruvate Carboxylase
To quantify pyruvate
carboxylase in various tissues, proteins in homogenates of tissues were
separated by SDS-gel electrophoresis and transferred to nitrocellulose,
which was then probed with I-streptavidin to detect
biotin-containing proteins by autoradiography. A protein with an M
of 116,000, identical to that of authentic
pyruvate carboxylase(11) , was present in pancreatic islets,
liver, and kidney, but heart, testis, and spleen contained little or
none of this protein (Fig. 1). These results are consistent with
what is known about the tissue distribution of the enzyme. Pyruvate
carboxylase enzyme activity is known to be abundant in liver and
kidney, which require the enzyme for gluconeogenesis, while many other
tissues, such as heart, are known to possess almost none of the enzyme (17, 18) . The density of the M 116,000 band from densitometric scans of a number of
autoradiographs of blots of homogenates from these tissues was averaged
and compared with a standard curve of authentic pyruvate carboxylase ( Fig. 1in Reference 5) to estimate the amount of pyruvate
carboxylase in the various tissues (Table 1). Islets contained 4
µg of pyruvate carboxylase/mg whole cell protein, while liver and
kidney contained 3.6 and 2.3 µg of the enzyme/mg of whole cell
protein. Table 1shows islets contain as much, if not more,
pyruvate carboxylase enzyme activity, as liver and kidney, as judged by
a CO fixation assay.
I-streptavidin. The abbreviations are as follows: PC, pure pyruvate carboxylase (0.3 µg); I,
islets; M, skeletal muscle; S, spleen); H,
heart; P, pancreas; L, liver; T, testis; K, kidney. The M
of the pyruvate
carboxylase band is 116,000, and that of the unidentified
biotin-containing protein(s) is about
80,000.
Malate Formation from Pyruvate by
Mitochondria
Pyruvate was added to mitochondria from islets and
other tissues, and the export of various metabolites to the medium was
measured. Mitochondria from islets, liver, and kidney, which contain
pyruvate carboxylase, exported a large amount of malate to the medium
and very little, if any, citrate, isocitrate, and aspartate, whereas
mitochondria from tissues that contain little or no pyruvate
carboxylase produced essentially no metabolites (testis formed a small
amount of citrate) (Figs. 2 and 3). The levels of these metabolites
inside the mitochondria from the non-islet tissues were measured and
were at or below the limit of detection for the assays (Fig. 2).
[1-
When islet
mitochondria were incubated with [1-C]PyruvateC]pyruvate,
radioactivity was exported form the mitochondria primarily as malate
(Figs. 4-6). The amount of radioactivity appearing in amino acids
was 3.9 ± 0.9% (mean ± S.E., n = 6) that
in malate and the radioactivity exported in citrate and isocitrate was
even lower and inconstant ( Fig. 5and Fig. 6). The amount
of radioactivity appearing in lactate was 0.5-3% of that
incorporated into malate (Fig. 4), indicating that pyridine
nucleotides, such as NAD, and enzymes from cytosol, such as lactate
dehydrogenase, contaminating the mitochondrial preparations were too
low to convert significant amounts of pyruvate into lactate making it
highly unlikely that C appearing in malate could have been
formed outside the mitochondria via the reverse reaction of malic
enzyme, a cytosolic enzyme, and cytosolic NADPH contaminating the
mitochondrial preparations. Therefore, any malate formed from pyruvate
must have been generated by pyruvate carboxylase in the mitochondrial
matrix.
C in malate from
pancreatic islet mitochondria given
[1-C]pyruvate. The mitochondrial pellet from
about 2,000 pancreatic islets was washed once and incubated in 170
µl of a mixture containing 2 mM
[1-C]pyruvate (24 mCi/mmol). Samples of 50
µl were removed every 10 min, centrifuged immediately, and the
medium removed from the mitochondrial pellet. An extract of the medium
was prepared as described under ``Experimental Procedures.''
Radioactivity in malate, citrate, isocitrate, and amino acids was
estimated by the enzymatic and column chromatographic method A.
Radioactivity in citrate and isocitrate was below or at the limit of
detection. The results of this experiment were confirmed by three
additional experiments (30-min time point
only).
C in malate,
citrate, and isocitrate from pancreatic islet mitochondria given
[1-C]pyruvate. The unwashed mitochondrial pellet
from about 2,000 pancreatic islets was incubated in 120 µl of
incubation medium containing 5 mM
[1-C]pyruvate (10 mCi/mmol) at 37 °C for 30
min, and radioactivity recovered in metabolites in the medium was
estimated by the enzymatic and column chromatographic method B as
described under ``Experimental Procedures.'' Results of three
experiments are shown in a bargraph. The absence of
a bar represents a value of zero.
C in malate from
pancreatic islet mitochondria given [1-14C]pyruvate.
The mitochondrial pellet from about 2,000 pancreatic islets was washed
once and incubated in 50 µl of a mixture containing 3.5 mM [1-C]pyruvate (28 mCi/mmol) for 30 min at
37 °C and centrifuged at 14,000 g for 1.5 min. The
medium was separated from the mitochondrial pellet, and and
radioactivity present in malate and lactate in the medium outside
mitochondria was identified by paper chromatography as described under
``Experimental Procedures.'' Radioactivity present in malate
was also estimated by the enzymatic and column chromatographic method
A. Results of two experiments are shown.
[U-
When intact islets were incubated with
[U-C]Succinate Dimethyl
EsterC]succinate dimethyl ester, radioactivity
appeared within the islets and surrounding medium in pyruvate and
lactate in three separate experiments (Table 2).
[1-CO,
[C]Malate, and Malate Formation by Islet
MitochondriaC]Pyruvate was incubated
with islet mitochondria in four separate experiments. The average of
the ratios of malate relative to CO formed was
1 (mean ± S.E. = 0.95 ± 0.2, n =
4), and the average of the ratios of the amount of C
appearing in malate relative to CO formed was
1.6 (mean ± S.E. = 1.6 ± 0.2, n =
4) (Fig. 7).
C incorporation into malate and
pyruvate decarboxylation as CO formation from
[1-C]pyruvate in pancreatic islet mitochondria.
The unwashed mitochondrial pellet from about 2,000 pancreatic islets
was incubated in 100 µl of incubation medium containing 5 mM [1-C]pyruvate (2 mCi/mmol) for 30 min at 37
°C. The reaction was stopped by adding perchloric acid, and the
evolved CO was measured. C
incorporated into malate was estimated by the enzymatic and column
chromatographic method B, and malate formed was measured
fluoremetrically as described under ``Experimental
Procedures.'' Results are from four separate experiments, and the
means ± S.E. are shown.
Pyruvate Malate Shuttle
Our recent studies of C flux from C-labeled glucose and methyl
succinate to CO in pancreatic islets with the CO ratios method indicated that about one-half
of glucose-derived pyruvate enters mitochondrial metabolism via
carboxylation and one-half via decarboxylation(1, 2) .
This amount of carboxylation clearly seems excessive, since citric acid
cycle intermediates, especially four-carbon dicarboxylic acids, should
accumulate to astronomical levels within the mitochondrial matrix and
probably interfere with mitochondrial metabolism, unless these
metabolites are continually removed. Because in any cell, utilization
of pyruvate carbon in the citric acid cycle equals that obtained via
decarboxylation of pyruvate (i.e. two carbons enter as
acetyl-CoA and two carbons are lost as CO in each turn of
the cycle), it follows that if intermediates are lost from the cycle,
there will be no oxaloacetate to condense with incoming acetyl-CoA and
the cycle will cease to operate. Since there is evidence for carbon
entering cycle intermediates via carboxylation in the pancreatic islet,
this suggests that carbon is entering the cycle to replenish carbon
that is exiting the cycle in this tissue.CO ratio
method(20) . In this method C-labeled substrates,
such as pyruvate and acetate (20) or glucose and methyl
succinate(1, 2) , which become citric acid cycle
intermediates, are added to cells. The ratio of pyruvate carboxylated
relative to that decarboxylated is calculated by comparing the loss of CO from inner carbons of cycle intermediates versus the loss of CO from the outer
carbons of cycle intermediates(20) . Outer carbons of succinate
are more likely to be lost with one turn of the cycle or in
decarboxylation reactions which branch from the cycle, such as the
reaction catalyzed by malic enzyme, whereas several turns of the cycle
are required to release inner carbons as CO. With
increasing carboxylation there is correspondingly less CO evolution from inner carbons of succinate,
because the inner carbons are more subject to dilution from unlabeled
carbon entering the cycle. The proposed shuttle in which malate is
continually recycled might provide a means for the beta cell to avoid
accumulating high amounts of four-carbon dicarboxylic acids and also
might explain the CO evolution patterns. Such
a shuttle is feasible because malic enzyme is present only in the
cytosol in the islet(3, 21) , as is the case with most
tissues, and pyruvate carboxylase is abundant in islet mitochondria.
CO, radioactivity is incorporated into malate,
fumarate, and citrate. In this early study it was not determined
whether these metabolites were recovered outside or inside the
mitochondria. However, in a recent study of dehydroepiandrosterone and
thermogenesis, this group showed that when pyruvate was applied to rat
liver mitochondria, malate and citrate were formed and 98% of these
metabolites were recovered in the medium outside the mitochondria (23) , which is in agreement with the results of the current
study (Fig. 2). This supported their hypothesis, suggested by
studies of dehydroepiandrosterone on the induction of malic enzyme and
other enzymes in liver, that ``malate formed from pyruvate in the
mitochondria can generate NADPH in the cytosol via malic enzyme'' (24) . It was further hypothesized that transhydrogenation
could occur via a series of reactions involving NADPH produced by malic
enzyme reducing dihydroxyacetone phosphate in the reaction catalyzed by
NAD-dependent glycerol phosphate dehydrogenase in the
cytosol(22, 23, 24) . The current study does
not address this overall scheme, but is focused only on obtaining
detailed evidence with metabolite assays and C flux for
the possible existence of a simple pyruvate malate shuttle in the
pancreatic islet. It has also been inferred from studies of drug
metabolism in liver that such a shuttle could provide NADPH for
reactions catalyzed by mixed function oxidases(25) .Mitochondrial Arm of the Pyruvate Malate
Shuttle
When given pyruvate, mitochondria from tissues, such as
pancreatic islets, liver, and kidney, which possess a large amount of
pyruvate carboxylase, export malate to the surrounding medium, whereas
the concentration of malate in the mitochondrial matrix remains
constant ( Fig. 2and Fig. 3). To further demonstrate the
mitochondrial arm of the proposed shuttle, pancreatic islet
mitochondria were incubated with [1-C]pyruvate
and the appearance of C in malate, citrate, isocitrate,
and amino acids in the surrounding medium was measured. Since carbon 1
of pyruvate is lost as CO in the pyruvate dehydrogenase
reaction, the only route by which radioactivity could appear in these
metabolites is via the reaction catalyzed by pyruvate carboxylase.
Estimates of C recovery in metabolites by three methods,
paper chromatography (Fig. 4) and two enzymatic and column
chromatographic methods ( Fig. 5and Fig. 6), showed that
radioactivity was exported from islet mitochondria predominantly as
malate with little radioactivity exported as amino acids and barely any
and inconsistently detectable radioactivity exported as citrate and
isocitrate.
Cytosolic Arm of the Pyruvate Malate Shuttle
To
demonstrate the possibility of the cytosolic arm of the shuttle,
[U-C]succinate dimethyl ester, a potent insulin
secretagogue(26, 27, 28, 29, 30, 31, 32, 33, 34) ,
was incubated with intact islets as a mitochondrial source of
four-carbon dicarboxylic acids. This compound is taken up by islets
where the methyl group is hydrolyzed leaving succinate to be
metabolized in the mitochondrion(1) . This experiment resulted
in the appearance of C in pyruvate and lactate both within
the islet and in the medium surrounding the islets (Table 2).
Since the pyruvate carboxylase reaction is, of course, essentially
irreversible, the decarboxylation of malate to pyruvate could have only
occurred via the malic enzyme reaction. It is noteworthy that carbon
from glutamine has been reported to appear in pyruvate in the islet (35) . Glutamine carbon would appear in pyruvate via glutamate,
-ketoglutarate, and four-carbon dicarboxylic acids within the
mitochondria and finally via the reaction catalyzed by malic enzyme.
The most potent insulin secretagogues are those that are metabolized
and there are very few potent metabolizable insulin secretagogues. The
ability to undergo conversion to pyruvate with formation of NADPH (see
below) may explain why methyl succinate and compounds that increase
glutamate metabolism (leucine and glutamine) (26, 28, 36, 37, 38) are
almost as potent insulin secretagogues as glucose.Carboxylation Versus Decarboxylation of Pyruvate in Islet
Mitochondria
The relatively high rate of carboxylation of
pyruvate previously observed in intact pancreatic islets (1, 2) was corroborated by experiments shown in Fig. 7in which CO production from islet
mitochondria metabolizing [1-C]pyruvate, an
estimate of pyruvate decarboxylation, was compared to both C incorporation into malate and to the total amount of
malate production estimated with a fluorometric metabolite assay, which
are estimates of pyruvate carboxylation. In four separate experiments
the ratio of malate to CO formed ranged from
0.5 to 1.6 (mean, 0.95) and the ratio of C appearing in
malate to CO formed ranged from 1.0 to 2.1
(mean, 1.6). These values agree with previous estimates obtained in
studies of intact pancreatic islets with the CO ratios method which indicated that the rate of pyruvate's
carboxylation was approximately equal to its decarboxylation
(35-65% carboxylation with an average of about 50% carboxylation; (1) and (2) ).Conclusions
The results of the experiments
described in this paper demonstrate the feasibility of a shuttle in
which pyruvate in the mitochondrion is carboxylated via pyruvate
carboxylase to oxaloacetate which is reduced to malate. Malate exits
the mitochondrion to the cytosol, where it is oxidatively
decarboxylated to pyruvate via the malic enzyme reaction. The pyruvate
then re-enters the mitochondrion with the net result being the
formation of NADPH in the cytosol (Fig. 8).-ketoglutarate via
isocitrate dehydrogenase in the cytosol. The -ketoglutarate could
directly enter the mitochondrion or undergo transamination with alanine
or aspartate to produce glutamate, which could enter the mitochondrion
and be metabolized or participate in a malate aspartate shuttle which
is believed to be present in the beta cell(10) .
)the protein disulfide
isomerase-thioredoxin
system(58, 59, 60, 61, 62) , nitrogen oxide synthase(63) , and quinone
reductase(64, 65) . The presence of the glutathione
reductase and thioredoxin-protein disulfide isomerase systems are
particularly interesting in the islet because of proposals that
sulfhydryl status of the islet is altered by insulin secretagogues (66, 67) .
)
I acknowledge the technical assistance of Carrie E.
Pomije, and thank Drs. Henry A. Lardy, Leonard A. Fahien, and Bernard
R. Landau for helpful discussion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 D. Lu, H. Mulder, P. Zhao, S. C. Burgess, M. V. Jensen, S. Kamzolova, C. B. Newgard, and A. D. Sherry 13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS) PNAS, March 5, 2002; 99(5): 2708 - 2713. [Abstract] [Full Text] [PDF]
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C. B. Wollheim and P. Maechler {beta}-Cell Mitochondria and Insulin Secretion: Messenger Role of Nucleotides and Metabolites Diabetes, February 1, 2002; 51(90001): S37 - 42. [Abstract] [Full Text] [PDF]
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T. K. Bratanova-Tochkova, H. Cheng, S. Daniel, S. Gunawardana, Y.-J. Liu, J. Mulvaney-Musa, T. Schermerhorn, S. G. Straub, H. Yajima, and G. W.G. Sharp Triggering and Augmentation Mechanisms, Granule Pools, and Biphasic Insulin Secretion Diabetes, February 1, 2002; 51(90001): S83 - 90. [Abstract] [Full Text] [PDF]
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S. G. Straub, H. Yajima, M. Komatsu, T. Aizawa, and G. W.G. Sharp The Effects of Cerulenin, an Inhibitor of Protein Acylation, on the Two Phases of Glucose-Stimulated Insulin Secretion Diabetes, February 1, 2002; 51(90001): S91 - 95. [Abstract] [Full Text] [PDF]
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C. V. Mobbs, L.-M. Kow, and X.-J. Yang Brain glucose-sensing mechanisms: ubiquitous silencing by aglycemia vs. hypothalamic neuroendocrine responses Am J Physiol Endocrinol Metab, October 1, 2001; 281(4): E649 - E654. [Abstract] [Full Text] [PDF]
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 P. Vernet, N. Fulton, C. Wallace, and R. J. Aitken Analysis of Reactive Oxygen Species Generating Systems in Rat Epididymal Spermatozoa Biol Reprod, October 1, 2001; 65(4): 1102 - 1113. [Abstract] [Full Text] [PDF]
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R. H. Skelly, B. Wicksteed, P. A. Antinozzi, and C. J. Rhodes Glycerol-Stimulated Proinsulin Biosynthesis in Isolated Pancreatic Rat Islets via Adenoviral-Induced Expression of Glycerol Kinase Is Mediated via Mitochondrial Metabolism Diabetes, August 1, 2001; 50(8): 1791 - 1798. [Abstract] [Full Text] [PDF]
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A.-D. Lajoix, H. Reggio, T. Chardès, S. Péraldi-Roux, F. Tribillac, M. Roye, S. Dietz, C. Broca, M. Manteghetti, G. Ribes, et al. A Neuronal Isoform of Nitric Oxide Synthase Expressed in Pancreatic {beta}-Cells Controls Insulin Secretion Diabetes, June 1, 2001; 50(6): 1311 - 1323. [Abstract] [Full Text]
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H. Makimura, T. M. Mizuno, X.-J. Yang, J. Silverstein, J. Beasley, and C. V. Mobbs Cerulenin Mimics Effects of Leptin on Metabolic Rate, Food Intake, and Body Weight Independent of the Melanocortin System, but Unlike Leptin, Cerulenin Fails to Block Neuroendocrine Effects of Fasting Diabetes, April 1, 2001; 50(4): 733 - 739. [Abstract] [Full Text]
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N. Lameloise, P. Muzzin, M. Prentki, and F. Assimacopoulos-Jeannet Uncoupling Protein 2: A Possible Link Between Fatty Acid Excess and Impaired Glucose-Induced Insulin Secretion? Diabetes, April 1, 2001; 50(4): 803 - 809. [Abstract] [Full Text]
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F. C. Schuit, P. Huypens, H. Heimberg, and D. G. Pipeleers Glucose Sensing in Pancreatic {beta}-Cells: A Model for the Study of Other Glucose-Regulated Cells in Gut, Pancreas, and Hypothalamus Diabetes, January 1, 2001; 50(1): 1 - 11. [Abstract] [Full Text]
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P. Maechler and C. B Wollheim Mitochondrial signals in glucose-stimulated insulin secretion in the beta cell J. Physiol., November 15, 2000; 529(1): 49 - 56. [Abstract] [Full Text] [PDF]
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 A. Schurr, R. S. Payne;, M. J. MacDonald, L. A. Fahien;, K. Eto, Y. Tsubamoto, and T. Kadowaki; NADH Shuttle and Insulin Secretion Science, February 11, 2000; 287(5455): 931a - 931. [Full Text]
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L. Segall, N. Lameloise, F. Assimacopoulos-Jeannet, E. Roche, P. Corkey, S. Thumelin, B. E. Corkey, and M. Prentki Lipid rather than glucose metabolism is implicated in altered insulin secretion caused by oleate in INS-1 cells Am J Physiol Endocrinol Metab, September 1, 1999; 277(3): E521 - E528. [Abstract] [Full Text] [PDF]
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G. G. Xu, Z.-y. Gao, P. D. Borge Jr., and B. A. Wolf Insulin Receptor Substrate 1-induced Inhibition of Endoplasmic Reticulum Ca2+ Uptake in beta -Cells. AUTOCRINE REGULATION OF INTRACELLULAR Ca2+ HOMEOSTASIS AND INSULIN SECRETION J. Biol. Chem., June 18, 1999; 274(25): 18067 - 18074. [Abstract] [Full Text] [PDF]
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S. Jitrapakdee, Q. Gong, M. J. MacDonald, and J. C. Wallace Regulation of Rat Pyruvate Carboxylase Gene Expression by Alternate Promoters during Development, in Genetically Obese Rats and in Insulin-secreting Cells. MULTIPLE TRANSCRIPTS WITH 5'-END HETEROGENEITY MODULATE TRANSLATION J. Biol. Chem., December 18, 1998; 273(51): 34422 - 34428. [Abstract] [Full Text] [PDF]
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A.-K. Parkkila, A. L. Scarim, S. Parkkila, A. Waheed, J. A. Corbett, and W. S. Sly Expression of Carbonic Anhydrase V in Pancreatic Beta Cells Suggests Role for Mitochondrial Carbonic Anhydrase in Insulin Secretion J. Biol. Chem., September 18, 1998; 273(38): 24620 - 24623. [Abstract] [Full Text] [PDF]
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 L. Zong-Chao, S. Efendic, R. Wibom, S. M. Abdel-Halim, C.-G. Ostenson, B. R. Landau, and A. Khan Glucose Metabolism in Goto-Kakizaki Rat Islets Endocrinology, June 1, 1998; 139(6): 2670 - 2675. [Abstract] [Full Text] [PDF]
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F. Schuit, A. De Vos, S. Farfari, K. Moens, D. Pipeleers, T. Brun, and M. Prentki Metabolic Fate of Glucose in Purified Islet Cells. GLUCOSE-REGULATED ANAPLEROSIS IN beta CELLS J. Biol. Chem., July 25, 1997; 272(30): 18572 - 18579. [Abstract] [Full Text] [PDF]
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R. J. Mertz, J. F. Worley III, B. Spencer, J. H. Johnson, and I. D. Dukes Activation of Stimulus-Secretion Coupling in Pancreatic beta-Cells by Specific Products of Glucose Metabolism J. Biol. Chem., March 1, 1996; 271(9): 4838 - 4845. [Abstract] [Full Text] [PDF]
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A. Khan, Z. C. Ling, and B. R. Landau Quantifying the Carboxylation of Pyruvate in Pancreatic Islets J. Biol. Chem., February 2, 1996; 271(5): 2539 - 2542. [Abstract] [Full Text] [PDF]
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M. J. MacDonald and L. A. Fahien Glutamate Is Not a Messenger in Insulin Secretion J. Biol. Chem., October 27, 2000; 275(44): 34025 - 34027. [Abstract] [Full Text] [PDF]
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