(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-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.
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-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.
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 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.
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 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.
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 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).
Figure 6:
Export of 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.
Figure 4:
Export of 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.
Figure 7:
Estimates of pyruvate carboxylation as
malate formation or 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.
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 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.
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 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) .
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 -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 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 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) .
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.
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