(Received for publication, November 2, 1995; and in revised form, December 15, 1995)
From the
The energy requirements of most cells supplied with glucose are
fulfilled by glycolytic and oxidative metabolism, yielding ATP. In
pancreatic -cells, a rise in cytosolic ATP is also a critical
signaling event, coupling closure of ATP-sensitive K
channels (K
) to insulin secretion via
depolarization-driven increases in intracellular Ca
([Ca
]
). We
report that glycolytic but not Krebs cycle metabolism of glucose is
critically involved in this signaling process. While inhibitors of
glycolysis suppressed glucose-stimulated insulin secretion, blockers of
pyruvate transport or Krebs cycle enzymes were without effect. While
pyruvate was metabolized in islets to the same extent as glucose, it
produced no stimulation of insulin secretion and did not block K
. A membrane-permeant analog, methyl pyruvate,
however, produced a block of K
, a sustained rise
in [Ca
]
, and an
increase in insulin secretion 6-fold the magnitude of that induced by
glucose. These results indicate that ATP derived from mitochondrial
pyruvate metabolism does not substantially contribute to the regulation
of K
responses to a glucose challenge,
supporting the notion of subcompartmentation of ATP within the
-cell. Supranormal stimulation of the Krebs cycle by methyl
pyruvate can, however, overwhelm intracellular partitioning of ATP and
thereby drive insulin secretion.
In pancreatic islets of Langerhans, glucose uptake by the
-cell initiates a cascade of events culminating in insulin
release. One of the key components in the insulin release process is
the generation of ATP from glucose metabolism(1, 2) .
Cytosolic ATP blocks ATP-sensitive K
(K
) (
)channels leading to
depolarization of the cell membrane, opening of voltage-gated
Ca
channels, and Ca
influx (3, 4) . Depolarization also induces release of
Ca
from endoplasmic reticulum (ER) stores, further
contributing to the elevation in intracellular calcium
([Ca
]
)(5, 6) .
Furthermore, the consequent Ca
depletion of the ER
activates a plasma membrane non-selective cationic channel that results
in further depolarization and enhancement of Ca
influx (7, 8, 9) . The interplay
between the ER Ca
stores and plasma membrane ion
channels underlies the glucose-stimulated coupling of membrane
potential and [Ca
]
oscillations (8, 9, 10, 11, 12) .
Insulin secretion results from these changes in
[Ca
]
in a process that
remains unclear.
A general principle of physiology holds that
metabolism and energy requirements are tightly coupled. Additionally,
the pancreatic -cell utilizes the precise measurement of its own
metabolism, via the degree of activation of K
,
to regulate insulin secretion. The dual role of metabolism in this cell
type necessitates discrete regulation of these specific functions. By
virtue of the fact that oxidative metabolism takes place in the
mitochondria, whereas the glycolytic enzymes are cytosolic or plasma
membrane-associated, compartmentation of metabolism can theoretically
occur, leading to functional intracellular microdomains of
ATP(13) .
While glycolysis in the -cell produces only
one-sixth of the total amount of ATP derived from the complete
oxidation of glucose(14) , the contribution of the much larger
mitochondrial Krebs cycle-derived ATP to
-cell glucose signaling
remains
controversial(15, 16, 17, 18) .
Recently, it has been reported that NADH derived from glycolysis and
processed into ATP by the operation of the transmitochondrial NADH
shuttles supplies the essential rise in cytosolic ATP necessary to
initiate block of K
and induce membrane
depolarization and [Ca
]
oscillations(19) . Using measurements of K
from single
-cells and
[Ca
]
and insulin
release from whole islets, we have further investigated the critical
steps in ATP production necessary for activation of stimulus-secretion
coupling. Our results suggest that glycolysis is critically involved in
glucose-stimulated insulin secretion, whereas the Krebs cycle appears
to play an insignificant role. The additional findings that a
membrane-permeant ester of pyruvate nonetheless is able to induce
insulin secretion indicate that ATP produced during the suprastimulated
operation of the Krebs cycle is able to initiate the
-cell
stimulus-secretion coupling signaling cascade but that in the normal
course of glucose metabolism this does not take place.
Islets of Langerhans were isolated from 3-7-month-old
C57BL/KsJ mice by collagenase digestion and cultured for 24 h for batch
islet insulin studies or for 2-10 days on glass coverslips for
[Ca]
studies, as
described elsewhere(5, 7, 8) . Single
-cells were dispersed as described
previously(7, 8) .
Through the subcellular localization of key enzymes,
glycolysis takes place in the cytosol, whereas the oxidation of the
glycolytic end product, pyruvate, takes place in the mitochondria.
While the plasma membrane is freely permeable to pyruvate, entry of the
carboxylic acid into the mitochondrion is regulated by a specific
transporter(24) . Fig. 1summarizes the relative effects
of glucose and pyruvate on whole-cell K recorded
from a single
-cell using perforated patch techniques. While
glucose caused a large suppression of K
,
pyruvate, at concentrations as high as 20 mM, had no effect.
These data are in agreement with the lack of stimulatory effect of
pyruvate on insulin secretion in rat islets (15) and on
[Ca
]
in mouse
islets(7) . Furthermore, the observations are consistent with
the lack of effect of the mitochondrial pyruvate transport inhibitor
-CHC on glucose-induced closure of K
in
mouse
-cells (18) or on glucose-stimulated elevations in
[Ca
]
in single HIT
cells(25) .
Figure 1:
Relative effects of
glucose, pyruvate, and methyl pyruvate on ATP-dependent
K currents. Shown are the membrane currents recorded
using perforated patch techniques from single pancreatic
-cells.
From a holding potential of -70 mV, alternating depolarizing and
hyperpolarizing voltage clamp steps of 160 ms duration and at a
frequency of 0.1 Hz were applied to -40 and -100 mV,
respectively. In control conditions, 2 mM glucose was present
in the bathing solution (trace marked control), the concentration of
glucose was increased to 12 mM (trace marked glucose), or 20
mM pyruvate or methyl pyruvate (traces marked pyruvate and MP,
respectively) were added. Shown are the mean changes in membrane
current (±S.E.) from five separate experiments. Note that
glucose and methyl pyruvate but not pyruvate itself were able to reduce K
current.
These observations suggested that, in the normal
course of events, either mitochondrial metabolism of glucose-derived
pyruvate to form ATP does not occur in -cells or, alternatively,
operation of the Krebs cycle does not contribute directly to the
initial steps in the glucose-dependent stimulus-secretion coupling
process. Consistent with either notion was the lack of effect of
3-nitropropionic acid, an inhibitor of the Krebs cycle enzyme succinic
dehydrogenase on glucose-induced closure of K
, (
)as well as the previously reported lack of effect of the
aconitase inhibitor monofluoroacetate(19) . On the other hand,
the membrane-permeant ester of pyruvic acid, methyl pyruvate, caused a
suppressive effect on K
equivalent to that of
glucose (Fig. 1). That this effect was not due to a direct block
of K
by methyl pyruvate is illustrated in Fig. 2. Using single channel recordings of K
from detached inside-out patches, where mitochondrial metabolism
of substrates to produce ATP can no longer take place, neither pyruvate
nor methyl pyruvate was able to block K
. On the
other hand, exogenously added ATP caused immediate channel closure (Fig. 2). These results suggested that the block of K
induced by methyl pyruvate was due to its
mitochondrial metabolism and resultant ATP generation.
Figure 2:
Methyl pyruvate does not directly block
single K channels in excised patches. Shown are
the effects of 20 mM pyruvate (upper right records)
and 20 mM methyl pyruvate (lower right records) on
single K
channel activity recorded in excised
patches from a
-cell in the inside-out configuration. Two channels
are present in the upper patch records, whereas the activity of three
channels is detectable in the lower records. Note that neither pyruvate
nor methyl pyruvate had any effect on single channel openings, whereas
ATP caused immediate suppression. The residual channel activity
recorded in the presence of ATP probably represents a single delayed
rectifier K
channel opening. The arrow head indicates the zero current level. The inset on the left shows summarized mean open channel probabilities (P
); when uncoupled from ATP production, methyl
pyruvate had no effect, whereas 2 mM ATP caused complete
suppression of channel openings.
We next
compared the relative effects of glucose, pyruvate, and methyl pyruvate
on alterations in cytoplasmic Ca in fura-2 loaded
mouse islets. Pyruvate, at concentrations up to 20 mM, had no
effect on [Ca
]
, again
consistent with the absence of effect of Krebs cycle processing of
glycolysis-derived substrates (Fig. 3). On the other hand, both
glucose and methyl pyruvate produced robust increases in
[Ca
]
. As reported previously,
12 mM glucose induced a triphasic alteration in
[Ca
]
: an initial decrease, due
to the activation of ER Ca
sequestration (phase
0)(7) ; a sustained transient related to ER Ca
release and secondary Ca
influx (phase
1)(5, 8, 9) ; and finally regular
oscillations, reflecting a complex interplay between the ER
Ca
store and Ca
entry through L-type Ca
channels (phase 2). It is of
interest that methyl pyruvate also induces an initial phase 0 (Fig. 3C), supporting the contention that the
activation of ER Ca
sequestration merely requires the
provision of ATP substrate for the ER Ca
ATPase(7) . On the other hand, methyl pyruvate never
induced [Ca
]
oscillations; at
lower concentrations (10 mM), a single
[Ca
]
transient was produced
instead of a sustained rise (see Fig. 11), supporting the notion
that a product of glycolysis was necessary to induce
[Ca
]
oscillations.
Figure 3:
Relative effects of glucose, pyruvate, and
methyl pyruvate on [Ca]
in single mouse islets. Shown are the effects of 12 mM glucose (A), 20 mM pyruvate (B), and 20
mM methyl pyruvate (C), added during the period
indicated by the filled bars, on fura-2 fluorescence measured
in single mouse islets. The data are expressed as the fluorescence
ratio measured at 340 and 380 nM, which is proportional to the
free cytosolic Ca
concentration. Prior to addition of
glucose, pyruvate, and methyl pyruvate, the islet was perfused with a
control Krebs-Ringer buffer containing 2 mM glucose. Note that
both glucose and methyl pyruvate produced robust alterations in
[Ca
]
, while pyruvate
gave an insignificant response.
Figure 11:
Dichloroacetate potentiates the
[Ca]
responses to
methyl pyruvate but not to glucose. A, lack of effect of 1
mM DCA on 12 mM glucose-stimulated (open
bar) [Ca
]
oscillations recorded in a single fura-2-loaded mouse islet. B, potentiation of methyl pyruvate-dependent elevations in
[Ca
]
by
dichloroacetate. Shown is a typical control response to 12 mM glucose (open bar), which after washout into a solution
containing 2 mM glucose was exposed to 10 mM methyl
pyruvate. This submaximal concentration of the methyl ester induced a
single [Ca
]
transient;
exposure to DCA (hatched bar) induced a second additional
[Ca
]
transient. C, pre-exposure to dichloroacetate potentiates methyl
pyruvate-induced elevations in
[Ca2+]
, with no effect on
glucose-stimulated alterations in
[Ca2+]
. Following 10 min
pre-exposure to 1 mM DCA (solid bar) in 2 mM glucose-containing external solution, addition of 12 mM glucose (open bar) produced a typical control response
with no evidence of potentiation. Following wash-off into 2 mM glucose but in the continued presence of dichloroacetate, 10
mM methyl pyruvate (cross-hatched bar) produced a
potentiated, sustained rise in
[Ca2+]
, typical of that produced by
20 mM of the methyl ester alone.
As final
evidence that the effects of methyl pyruvate on K and [Ca
]
were due to
stimulation of mitochondrial ATP production, we examined the effects of
sodium azide, an inhibitor of mitochondrial oxidative
phosphorylation(26) . Following exposure to 3 mM sodium azide, an effective inhibitory concentration in
-cells(26) , methyl pyruvate produced a greatly attenuated
[Ca
]
rise, an effect that was
reversible following sodium azide removal (Fig. 4). In addition,
the sustained elevation of [Ca
]
induced by 20 mM methyl pyruvate was reversed by
addition of sodium azide (Fig. 4).
Figure 4:
Inhibition of mitochondrial oxidative
phosphorylation ablates
[Ca]
-elevating effects
of methyl pyruvate. In the upper panel, a short 12 mM glucose stimulus (filled bar) is shown in a fura-2 loaded
single mouse islet. After washout with a 2 mM glucose-containing external solution, the islet was then exposed
to 3 mM sodium azide (cross-hatched bar), which
caused an elevation in
[Ca
]
, most likely due
to leakage of Ca
from the poisoned mitochondria.
Sequential exposure to 20 mM methyl pyruvate (labeled MP, open bar) produced a greatly attenuated rise in
[Ca
]
. Following a
10-min washout period of the azide and methyl pyruvate, second exposure
to 20 mM methyl pyruvate then elicited a full
[Ca
]
response,
indicating that the effects of sodium azide are reversible and not
associated with nonspecific impairment of islet responsiveness. In the lower panel, the sequence of additions was reversed; following
a robust [Ca
]
response
to 20 mM methyl pyruvate, exposure to sodium azide in the
continued presence of methyl pyruvate produced an almost complete
ablation of the methyl pyruvate-activated
[Ca
]
rise. Again,
following washout of sodium azide, responsiveness to a 12 mM glucose challenge was immediately
restored.
It would seem, therefore,
that glycolytic but not Krebs cycle processing of glucose-derived
substrates was necessary to initiate changes in K channel activity and
[Ca
]
, prerequisites for
downstream initiation of insulin secretion. Nevertheless, a
membrane-permeant analog of pyruvate was able to largely reproduce the
effects of glucose on K
and
[Ca
]
by stimulating
mitochondrial ATP synthesis.
At physiological concentrations, both
glucose and the early glycolytic intermediate, D-glyceraldehyde, stimulate insulin synthesis and secretion in
whole islets(24, 27) . Iodoacetate (IAA) inhibits
glyceraldehyde phosphate dehydrogenase, blocking glycolysis and halting
further glucose oxidation prior to the production of NADH and
ATP(28) . When islets were exposed to 1 mM IAA, there
was a 78% inhibition of glucose-stimulated insulin secretion, compared
to control (Fig. 5), consistent with earlier findings of the
suppressive effects of IAA on proximal steps in the -cell glucose
stimulus-secretion coupling cascade(19) . Basal insulin
secretion in the presence of 2 mM glucose was, however,
elevated by exposure to IAA (Fig. 5). Since this effect was only
seen in low glucose, it is unlikely to be a consequence of the net
depletion of ATP produced by the unaffected proximal phosphorylation
steps in glycolysis. An alternative explanation is that in a manner
similar to the related glyceraldehyde phosphate dehydrogenase inhibitor
iodoacetamide, low concentrations (0.5 mM) might activate
glyceraldehyde phosphate dehydrogenase, especially at lower glucose
concentrations(29) .
Figure 5: Inhibition of glycolysis reduces, whereas inhibition of pyruvate utilization has no effect on glucose-stimulated insulin secretion. Shown are the insulin secretion responses to low (2 mM glucose, filled bars) and high (12 mM glucose, cross-hatched bars) glucose exposure in the absence and presence of a series of inhibitors of glycolysis and pyruvate transport. These include IAA, an inhibitor of glyceraldehyde-3-dehydrogenase, arsenate, an uncoupler of glycolytic ATP production, and CHC, an inhibitor of pyruvate transport. Note that arsenate and IAA both inhibited glucose-stimulated insulin secretion; since IAA halts glycolysis but arsenate only uncouples glycolytic ATP production and does not therefore prevent downstream metabolism of pyruvate, this result suggests that glycolysis alone plays a critical role in glucose-stimulated insulin secretion. On the other hand, CHC had a mild potentiating effect on glucose-stimulated insulin secretion, an effect also observable following the combined addition of arsenate and CHC. These observations with CHC may reflect the preferential conversion of pyruvate to lactate under conditions of impaired mitochondrial pyruvate transport, with enhanced reformation of NAD to prime the residual production of glycolytic ATP still taking place in the presence of the inhibitors.
Glycolysis produces ATP directly
through dephosphorylation reactions catalyzed by the actions of
phosphoglycerate kinase on 1,3-diphosphoglycerate and pyruvate kinase
on phosphoenolpyruvate(14) . To investigate the role played by
these ATPs on insulin secretion, we utilized arsenate, which uncouples
ATP production by phosphoglycerate kinase without halting
glycolysis(30) . In the presence of 1 mM arsenate,
there was a 70% reduction in glucose-stimulated insulin secretion (Fig. 5). We previously reported that arsenate pretreatment had
no effect on glucose-induced closure of K(19) . However, the insulin secretion
process, unlike the early steps in the glucose signaling pathway, is an
energy-consuming process and may therefore be expected to be more
sensitive to small changes in ATP production. On the other hand,
prolonged exposure to arsenate is known to inhibit mitochondrial
oxidative phosphorylation(31) , so the different results
obtained in the insulin secretion experiment (90 min exposure) and K
measurements (10-20 min exposure) may
reflect this fact. However, in contrast to the effects of IAA, there
was no increase in basal insulin secretion (Fig. 5). Finally,
exposure to 1 mM sodium fluoride, an inhibitor of enolase,
which catalyzes the formation of the pyruvate precursor,
phosphoenolpyruvate (14) , reduced glucose-stimulated insulin
secretion from 367 ± 80 to 265 ± 85 pM/islet/h
(mean ± S.E., n = 3) with no effect on basal (2
mM glucose) insulin secretion (18 ± 3 versus 23 ± 6). These results emphasize the importance of
glycolytic ATP in the insulin release process and suggest that a small
decrease in ATP production has a marked affect on insulin secretion.
The second source of glycolytic ATP is generated indirectly through
the mitochondrial processing of NADH derived from the oxidation of
glyceraldehyde-3-phosphate. The NADH reducing equivalents must cross
the mitochondrial membrane to generate ATP through oxidative
phosphorylation. This is accomplished by means of two electron shuttle
systems: the malate-aspartate shuttle and the glycerol phosphate
shuttle, which deposit their electrons at sites 1 and 2 of the electron
transport chain, respectively(19, 32) . Rotenone and
antimycin A block these respective sites (19, 33, 34) and were previously found to
inhibit a glucose-dependent block of K and
elevations in [Ca
]
in mouse
pancreatic
-cells and islets, respectively(19) . Rotenone
(50 nM) caused a 63% inhibition of insulin release, and
antimycin A (200 nM) caused a 59% inhibition of insulin
release in 12 mM glucose-stimulated islets compared to control (Fig. 6).
Figure 6: Inhibition of mitochondrial electron transport inhibits glucose-dependent insulin secretion. Shown are the insulin secretion responses to low (2 mM glucose, filled bars) and high (12 mM glucose, cross-hatched bars) glucose exposure in the absence and presence of inhibitors of site 1 and site 2 mitochondrial electron transport, antimycin A and rotenone, respectively. Note that both agents specifically inhibited glucose-stimulated insulin secretion, with no significant effect on basal (2 mM glucose-stimulated) secretion. These data support the role of the transmitochondrial NADH shuttles in processing glycolytically generated NADH into ATP.
Our findings with rotenone support the suggested
importance of the malate-aspartate shuttle in
islets(32, 35) . Aminooxyacetate inhibits the
malate-aspartate shuttle by suppressing aspartate aminotransferase (36) . Consistent with this action, 5 mM aminooxyacetate produced a 53% inhibition of insulin secretion in
the presence of 12 mM glucose, from 371 ± 103 to 176
± 34 pM/islet/h (mean ± S.E., n = 3, p < 0.05), with no effect on basal insulin
release (13 ± 2 versus 12 ± 2
pM/islet/h). As far as the alternative NADH shuttle is
concerned, mitochondrial glycerol phosphate dehydrogenase activity is
60-fold greater in the islet than in the liver and 10-fold greater in
-cells than in non-
-islet cells (37) . To further
investigate the significance of the operation of this shuttle to
glucose-stimulated insulin secretion, we used
2-aminobicyclo[2,2,2]heptane-2-carboxylate, which reportedly
increases the flux through the glycerol phosphate shuttle most likely
by a direct activation of the FAD-linked mitochondrial glycerophosphate
dehydrogenase(38) . In the presence of 12 mM glucose,
5 mM 2-aminobicyclo[2,2,2]heptane-2-carboxylate
produced a 76% stimulation of insulin secretion over control (412
± 69 versus 230 ± 16 pM/islet/h, n = 3, p < 0.02) and no significant change in
basal release (34 ± 12 versus 12 ± 9
pM/islet/h, n = 3). These data suggest that a
significant contribution is played by glycolytic NADH-derived ATP in
the regulation of glucose-dependent insulin secretion even though it
only contributes one-fifth the number of reduced equivalents produced
during mitochondrial oxidation of glucose-derived substrates.
To
investigate the contribution of pyruvate-derived ATP to the insulin
secretion response in glucose-stimulated islets, we blocked pyruvate
utilization with -CHC. In accordance with our other findings,
exposure to 1 mM
-CHC had no suppressive effect on basal
or glucose-stimulated insulin secretion (Fig. 7); in fact, a
slight stimulatory effect in the presence of 12 mM glucose was
often seen. Monofluoroacetate blocks the action of the Krebs cycle
enzyme aconitase on citrate(19, 39) , halting the
Krebs cycle prior to the production of reduced nucleotides or GTP.
Exposure of islets to 2 mM monofluoroacetate had no effect on
basal or glucose-stimulated insulin secretion (Fig. 7),
suggesting that production of ATP by the Krebs cycle is not
significantly contributing to the insulin response to glucose.
Figure 7: Absence of effect of Krebs cycle inhibition on glucose-stimulated insulin secretion. Shown are the insulin secretion responses to low (2 mM glucose, filled bars) and high (12 mM glucose, cross-hatched bars) glucose exposure in the absence and presence of monofluoroacetate, an inhibitor of the Krebs cycle enzyme aconitase, and CHC, an inhibitor of mitochondrial pyruvate transport. In neither case was an inhibition of glucose-stimulated insulin secretion detectable.
Despite these negative data relating to the importance of Krebs
cycle-derived ATP to glucose-dependent insulin secretion, 20 mM methyl pyruvate produced a 6-fold greater stimulation of insulin
release over controls in 12 mM glucose (Fig. 8). An
equivalent stimulation of insulin secretion by methyl pyruvate was
observed in the presence of 2 or 12 mM glucose, suggesting
that the secretory process was maximally stimulated by the pyruvate
ester. Furthermore, addition of glucose in the presence of methyl
pyruvate did not further elevate [Ca]
(Fig. 9). The potent secretagogue and
[Ca
]
-elevating effects of
methyl pyruvate suggested that the pyruvate ester had provided the
-cell access to a large source of ATP unavailable through normal
glucose oxidation. Mono and dimethyl esters of the Krebs cycle
substrate, succinate, also stimulate insulin
release(40, 41) , while succinate itself is
ineffective(40) .
Figure 8: Methyl pyruvate augments insulin secretion in a glucose-independent manner via a mechanism requiring intramitochondrial liberation of pyruvate. Shown are the insulin secretion responses to low (2 mM glucose, filled bars) and high (12 mM glucose, cross-hatched bars) glucose exposure in the absence and presence of 20 mM methyl pyruvate (MP). Methyl pyruvate caused a 6-fold increase in insulin secretion, an effect that was independent of the bathing glucose concentration, indicating maximal stimulation. Addition of CHC, an inhibitor of mitochondrial pyruvate transport, had no effect on the insulinotropic effects of methyl pyruvate, suggesting that only intramitochondrial liberation of free pyruvate could produce the observed effects.
Figure 9:
Methyl pyruvate induces changes in
[Ca]
similar to those
produced by high glucose that require intramitochondrial pyruvate
generation. A, shown are the effects on
[Ca
]
in fura-2-loaded
mouse islets of 20 mM glucose (open bar) and 20
mM methyl pyruvate (filled bar). Note that 20 mM glucose, in contrast to the effects of 12 mM glucose (see Fig. 3) induced a sustained rise in
[Ca
]
preceded by a
transient fall. 20 mM methyl pyruvate reproduced this response
in an identical fashion, and exposure to 12 mM glucose in the
continued presence of methyl pyruvate did not cause an additional rise
in [Ca
]
. B.
Addition of 1 mM CHC, an inhibitor of mitochondrial pyruvate
transport, prolonged the duration of the initial fall in
[Ca
]
induced by methyl
pyruvate but otherwise had no effect on the subsequent
[Ca
]
, indicating that
intra-mitochondrial liberation of free pyruvate was required to produce
the observed changes in
[Ca
]
.
It seemed unlikely that the esterified
form of pyruvate would be a more favorable metabolic substrate than
pyruvate itself. Since -cells are freely permeable to
pyruvate(42, 43) , it is also unlikely that methyl
pyruvate stimulation results from an increase in cytosolic,
de-esterified pyruvate. It seemed more probable that methyl pyruvate
traversed the mitochondrial membrane, circumventing the pyruvate
transporter, and was then de-esterified, liberating pyruvate at its
site of oxidation. To test this hypothesis, we measured the effects of
methyl pyruvate on islets in the presence of 1 mM
-CHC.
The mitochondrial transport inhibitor had no effect on
[Ca
]
response to 20 mM methyl pyruvate (Fig. 9). Furthermore,
-CHC exposure
did not alter the insulin secretory responses to methyl pyruvate (Fig. 8), indicating that mitochondrial, and not cytosolic
de-esterification, enables methyl pyruvate to produce its effects on
[Ca
]
and insulin secretion.
To confirm that the potent insulin secretagogue effects of the
pyruvate ester were due to the delivery of pyruvate to the site of
action of Krebs cycle enzymes within the mitochondria, we tested the
effects of dichloroacetate (DCA), a well characterized activator of the
pyruvate dehydrogenase enzyme complex(44, 45) . In the
presence of a submaximal concentration of methyl pyruvate, insulin
secretion was augmented by 33% by DCA (Fig. 10). On the other
hand, the presence of 1 mM DCA produced no appreciable
increase in glucose-stimulated insulin secretion (Fig. 10). This
lack of potentiating effect of DCA on glucose-dependent insulin
secretion from mouse islets is in agreement with a previous report
using rat islets (15) and indicates that the pyruvate produced
during glucose metabolism is largely ineffective in further augmenting
-cell stimulation-secretion coupling processes. We attempted to
correlate the methyl pyruvate-specific stimulation of insulin secretion
by DCA with alterations in
[Ca
]
. Addition of 1 mM DCA in the presence of 2 or 12 mM glucose had no
consistent effect on [Ca
]
(Fig. 11A). However, in the presence of a
submaximal concentration of methyl pyruvate (10 mM), which
itself activated a single [Ca
]
transient, addition of DCA consistently produced an additional
[Ca
]
transient (Fig. 11B). Pre-exposure to DCA converted the single
[Ca
]
transient typically
produced by 10 mM methyl pyruvate into a sustained rise,
typical of a response normally produced by 20 mM methyl
pyruvate (Fig. 11C). Taken together with the earlier
findings of a block of K
by methyl pyruvate,
these results reinforce the view that minimal utilization of
glucose-derived pyruvate for initiation of the stimulus-secretion
coupling cascade occurs in
-cells and that circumvention of
pyruvate transportation using a membrane-permeable ester provides
access to a huge ATP-generating and insulin secretory capacity.
Figure 10: Methyl pyruvate, but not glucose-stimulated insulin secretion, is potentiated by activation of the Krebs cycle. Shown are the insulin secretion responses to low (2 mM glucose, filled bars) and high (12 mM glucose, cross-hatched bars) glucose exposure in the absence and presence of 1 mM DCA, an activator of the pyruvate dehydrogenase complex. Also shown are the effects of DCA on the secretagogue activity of 10 mM methyl pyruvate (MP). Note that while the methyl pyruvate response was potentiated by dichloroacetic acid, there was no effect on glucose-stimulated insulin secretion.
The
observations with methyl pyruvate are not consistent with previous
reports on the lack of an insulinotropic effect of pyruvate. Exposure
of islets to 30 mM pyruvate produced intracellular levels of
pyruvate 5-fold higher than those found following exposure to 16.7
mM glucose(15) . Despite this, pyruvate did not
initiate insulin release (15, 17) and failed to show a
significant increase in NADH or ATP levels, even though pyruvate
oxidation was taking place (15) . We considered whether the
discrepancy between the effects of glucose and pyruvate were due to
differing rates of metabolism. Consistent with previous
reports(15) , we found on the basis of changes in pH detected
using a microphysiometer (Fig. 12) or direct measurements of CO
production from
[U-
C]pyruvate that pyruvate was oxidized as
effectively as glucose. Thus, 20 mM of
[U-
C]pyruvate yielded 51 pmol of
CO
/h/islet, comparable to rates of oxidation
measured with glucose(22) . Incubation with 1 mM
-CHC reduced the pyruvate oxidation rate to 8 pmol/h/islet,
indicating that the concentration of the pyruvate transport inhibitor
utilized in the K
,
[Ca
]
, and insulin secretion
assays was sufficient to block pyruvate transport and was in agreement
with other findings demonstrating inhibition by
-CHC of
CO
release from labeled pyruvate in HIT-T15
insulinoma cells(25) .
Figure 12:
Pyruvate is effectively oxidized in
islets and induces changes in metabolism. The upper panel demonstrates, using [U-C]pyruvate, that
significant oxidation of the Krebs cycle substrate takes place in rat
islets, an effect that was substantially attenuated by 1 mM
-CHC, an inhibitor of mitochondrial pyruvate transport. The lower panel shows the relative effects of 20 mM pyruvate (hatched bar) and 12 mM glucose (solid bar) on rat islet metabolism as assessed by changes in
extracellular pH using a microphysiometer.
The discrepancy between the rates of
pyruvate oxidation and lack of ATP signaling and an allied
insulinotropic effect is puzzling but has several explanations. First,
some cytosolic pyruvate may be shunted off into lactate, thereby
consuming reduced nucleotides destined for ATP production in the
process. Second, pyruvate preferentially inhibits the oxidation of
endogenous nutrients, reducing the apparent yield of ATP compared to
glucose(15) . Indeed, when correcting for such phenomena, the
net generation of reducing equivalents by glucose and pyruvate became
strictly proportional to their secretagogue activities(15) .
Yet another explanation is that the results could reflect the oxidation
of pyruvate by non--cells in the whole islets used, which would
suggest that measurement of pyruvate oxidation as it applies to islet
-cells is overstated. This controversy as to whether pyruvate is
actually metabolized in
-cells has, however, been recently
resolved by the demonstration in cell-sorted rat
-cells of
efficient mitochondrial pyruvate oxidation as well as oxidation of
acetyl-CoA in the Krebs cycle(46) .
The contrastingly potent
secretagogue effects of pyruvate in HIT insulinoma cells (25) indicates that they differ markedly from normal
-cells in their pyruvate metabolism pathways. Indeed, the
hypersensitivity of HIT cells to glucose (4) may reflect the
more efficient utilization of glucose-derived pyruvate for ATP
generation and stimulation of insulin secretion or altered expression
of key enzymes involved in glucose metabolism. For instance, islets
lack phosphoenolpyruvate carboxykinase(47, 48) , an
enzyme which permits pyruvate to re-enter glycolysis. When Escherichia coli-derived phosphoenolpyruvate carboxykinase is
expressed in
-cells, pyruvate becomes capable of promoting insulin
synthesis (49) . This further supports the singular importance
of glycolysis in regulating glucose-stimulated signal transduction in
-cells.
Our findings with methyl pyruvate suggest that when pyruvate is delivered directly to the mitochondria, a large stimulation of ATP production and insulin secretion takes place. Assuming that de-esterification of the methyl pyruvate primarily takes place in the mitochondrion, then certain metabolic costs associated with the processing of glucose-derived pyruvate will be saved. Thus, loss of substrate by conversion to lactate will not occur, since this process only takes place in the cytosol. Furthermore, the energy expended in operating the mitochondrial pyruvate transporter will also be saved. The question remains, however, whether this is an adequate enough explanation to reconcile the contrasting effects of pyruvate and methyl pyruvate.
It is important to distinguish at this point the dual
effects of ATP in -cells. Clearly, ATP is an important regulator
of insulin secretion by virtue of its critical role in initiating the
stimulus-secretion cascade by blocking K
channels. Additionally, the processing of ATP is necessary to
fuel the energy-consuming aspects of the insulin secretion process.
These disparate roles of ATP in the
-cell can explain the
divergent effects on metabolism and secretion of some of the agents
that we have employed. Thus, while pyruvate is ineffective in
initiating insulin secretion by failing to inhibit K
channels (Fig. 1), it is able to potentiate
glucose-stimulated insulin secretion(15) , presumably by
supplementing ATP destined for energy-consuming purposes. By the same
token, the inhibitory effects of arsenate on insulin secretion are
explicable on the basis of depleting ATP destined for energy
requirements without the impairing glucose-induced block of K
( (19) and Fig. 5).
Thus, an
alternative solution for the differing importance of glycolytic and
Krebs cycle processing of glucose-derived metabolites in -cell
signal transduction depends on the concept of functional
compartmentation of ATP production. There are numerous reports of cells
taking advantage of the compartmentalization of ATP production for
signaling purposes(13, 50) . For instance, Weiss and
colleagues (51, 52) have amply demonstrated a similar
dependence on glycolysis for regulation of K
in
cardiac myocytes and a close physical association between functional
glycolytic enzymes and these channels(51) . Another report
indicates that glycolytic and oxidative ATP account for different
phases of K
clearance in some neurons (53) and
that different types of muscle fibers rely specifically on glycolytic
or oxidative metabolism(54) . Another manifestation of
intracellular compartmentation of ATP production could be achieved if
there was heterogeneity of mitochondria. Mitochondrial heteroplasmy has
been suggested to underlie a number of heritable diseases, and the
evidence for functionally different mitochondria being present within a
single cell type is
accumulating(55, 56, 57, 58, 59) .
For instance, one recent report supports the existence of altered
pyruvate carboxylase expression within mitochondria of the same cell
resulting in completely different Krebs cycle operations(60) .
Thus, it is conceivable that mitochondria within the
-cell might
have different capacities for utilizing pyruvate and that with a
restricted subcellular distribution could result in the preferential
role of non-Krebs cycle-dependent glucose metabolism in the regulation
of insulin secretion.
The potent effects of methyl pyruvate may
result from its concentration in the mitochondria. It is known that
free acids formed from esterified precursors, for instance the
acetomethoxy ester of fura-2, can accumulate in intracellular
compartments at concentrations several orders of magnitude higher than
the bathing ester concentration(61) . This occurs by virtue of
the large concentration gradient for the esterified form created by the
continual de-esterification by esterases present in the intracellular
compartment and the consequent trapping of the free acid. Thus,
pyruvate would be expected to accumulate to extremely high levels in
mitochondria and cause such an over-stimulation of ATP production that
the cell would be flooded with ATP, overcoming any functional
microdomains of ATP that might exist and directly interacting with
plasma membrane-localized K. By contrast, the
transport of glycolytically produced pyruvate would be rate-limited by
the operation of the mitochondrial transporters as well as being
subject to shunting off to form lactate. The large stimulation of
insulin secretion produced by methyl pyruvate supports this hypothesis;
on the basis of molar equivalents, methyl pyruvate produced a 6-fold
greater increase in insulin release than glucose. Direct evidence to
support enhanced Krebs cycle ATP production is, however, lacking.
Attempts to measure [U-
C]methyl pyruvate
oxidation were unsuccessful as the ester was too unstable to allow
reliable measurements to be made. In the future, experiments utilizing
oxygen-burst measurements in islets with methyl pyruvate may be able to
confirm this proposition.
In conclusion, increases in glycolytic ATP
alone are primarily responsible for glucose-dependent insulin
secretion, although this pool accounts for only one-sixth of the total
ATP that can be produced by the complete oxidation of glucose. Our
results with -CHC and DCA on glucose- and methyl pyruvate-induced
stimulation of insulin secretion further suggest that the
-cell
does not fully utilize the ATP-generating capabilities that would
follow from the complete metabolism of glucose. The
-cell may be
taking advantage of the compartmentalization of ATP production,
utilizing glycolytic ATP to regulate K
and the
following downstream events. The
-cell's primary role is to
sense the amount of glucose present and to respond by releasing an
appropriate amount of insulin. Where sensing of glucose is important,
glycolysis is no doubt capable of producing a measured rise in ATP. If
this sensing system needed to be fine tuned enough to detect small
changes in glucose concentration coupled to a suitable insulin
response, large amounts of ATP from the complete oxidation of glucose
might well overwhelm that sensing system. The disadvantage, however, of
such a singular dependence of the
-cell on glycolysis-derived ATP
for glucose signaling is the inherent loss of signal plasticity. This
heightens the likelihood that minor defects in glycolytic processing
may translate into a
-cell-specific breakdown in glucose sensing,
a hallmark of non-insulin-dependent diabetes mellitus.