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(Received for publication, July 15, 1996, and in revised form, November 8, 1996)
From the Yale University School of Nursing, New Haven, Connecticut
06536-0740
ABSTRACT The methyl ester of pyruvic acid (methyl
pyruvate) stimulated a dose-dependent increase in insulin
secretion from isolated perifused rat islets. The threshold level for
release was about 10 mM, and at 20 mM the
addition of MP to perifused islets resulted in a large first phase of
secretion followed by an insulin-secretory response that was sustained
for at least 40 min. When compared to the effects of 20 mM
glucose, peak first-phase release rates in response to 20 mM methyl pyruvate were comparable, but the second phase of
release was only about 10-15% of that observed with an equimolar
level of the hexose. The stimulatory effects of 20 mM
methyl pyruvate on secretion were abolished by the K1+-ATP
channel blocker diazoxide (200 µM) and by the calcium
channel antagonist nitrendipine (500 nM). The glucokinase
inhibitor mannoheptulose (20 mM) had no adverse effect on
the secretory response to 20 mM methyl pyruvate, whereas 10 µM forskolin amplified the insulinotropic action of MP.
Sodium pyruvate alone or in combination with 10 µM
forskolin had no insulinotropic effect. In additional experiments islet
phosphoinositide pools were labeled with
myo-2-[3H]inositol, and the subsequent
accumulation of labeled inositol phosphates was used to monitor the
activation of phospholipase C. Methyl pyruvate stimulated a
dose-dependent increase in inositol phosphate levels when
measured after a 30-min incubation period with a maximal increase of
about 300% at 20 mM methyl pyruvate. The increase in
phosphoinositide hydrolysis caused by methyl pyruvate (20 mM) was, like insulin secretion, reduced by both diazoxide and nitrendipine but was immune to inhibition by mannoheptulose. Pyruvate (20 mM) had no effect on inositol phosphate
accumulation. Prior short-term exposure to methyl pyruvate sensitized
islets to subsequent stimulation with 15 mM glucose. Sodium
pyruvate did not sensitize islets. These findings support the concept
that the mitochondrial metabolism of nutrient molecules is an event sufficient to acutely augment insulin release from the beta cell, to
increase phospholipase C-mediated phosphoinositide hydrolysis, and to
induce time-dependent potentiation of insulin
secretion.
INTRODUCTION The regulation of fuel-induced insulin secretion from pancreatic
beta cells depends upon the intermediary metabolism of these compounds
via several established metabolic pathways (1). In addition to glucose,
mannose, glyceraldehyde, and dihydroxyacetone, which are metabolized
initially by cytosolic glycolytic enzymes (2-5), a variety of
insulinotropic nutrient molecules are metabolized solely within the
mitochondria. These include among others leucine, monomethylsuccinate,
and Standing at odds with this unifying concept, implicating the importance
of mitochondrial signals in the regulation of secretion regardless of
the nutrient molecule used, are studies with pyruvate. Although it
seems to be well metabolized, pyruvate alone has no insulinotropic
effect, although in combination with stimulatory glucose, a small
stimulatory action has been described (16). Most recently, the
methylated derivative of pyruvate has been reported to augment the
release of insulin from the beta cells of cultured mouse islets (17).
Thus, methylation seems to confer upon pyruvate stimulatory actions not
inherent in the unaltered pyruvate molecule. This situation is similar
to the effects of methylation on the succinate molecule, which alone
has no insulinotropic effect but when methylated now exhibits many of
the same stimulatory characteristics as glucose (7, 10, 18). Presumably
methylation renders succinate, and perhaps pyruvate as well, more
membrane-permeable and allows higher levels of the compound to be
achieved and acted upon by mitochondrial enzymes. In the present series
of studies we explored in more detail the effects of methyl pyruvate
(MP) on the beta cell. The dose dependence and calcium dependence of the insulinotropic effects of MP, the kinetics of insulin secretion from freshly isolated and perifused rat islets, as well as the ability
of MP to influence PI hydrolysis in
[3H]inositol-prelabeled islets were determined. The
findings support the view that mitochondrial metabolism of methyl
pyruvate is an event sufficient to generate the necessary metabolic and
second messenger molecules to activate the beta cell insulin-secretory apparatus in a manner comparable in some regards to that seen with
glucose. From a quantitative perspective, however, the maximal effects
of MP on second-phase release from rat islets are significantly less
than those observed with equimolar glucose.
MATERIALS AND METHODS The detailed methodologies used to assess insulin output from
collagenase-isolated islets have been described previously (19, 20).
Male Sprague-Dawley rats were used in all studies. All animals were
treated in a manner that complied with the National Institutes of
Health Guidelines for the Care and Use of Laboratory Animals. The
animals were fed ad libitum and weighed 300-450 g. After
Nembutal (pentobarbital sodium, 50 mg/kg; Abbott)-induced anesthesia
islets were isolated by collagenase digestion and handpicked using a
glass loop pipette under a stereo microscope. They were free of
exocrine contamination. After isolation some groups of islets were
directly perifused in a Krebs-Ringer bicarbonate (KRB) buffer at a flow
rate of 1 ml/min. Perifusate solutions were gassed with 95%
O2, 5% CO2 and maintained at 37 °C.
Perifusate samples were collected at appropriate times and analyzed for
insulin content. To label PI pools, other groups of 18-22 freshly
isolated islets were placed in small glass vials and incubated for
3 h in a myo-[2-3H]inositol-containing
KRB solution made up as follows. 10 µCi of
myo-[2-3H]inositol (specific activity, 16-23
Ci/mmol) were placed in a 10 mm × 75-mm culture tube. To this
aliquot of label 250 µl of warmed (to 37 °C) and oxygenated KRB
medium supplemented with 5 mM glucose were added. After
mixing, 240 µl of this were gently added to the vial with islets. The
vial was capped with a rubber stopper, gassed for 10 s with 95%
O2, 5% CO2, and incubated at 37 °C. After
90 min the vials were again gently oxygenated. After the labeling
period, the islets were washed with 5 ml of fresh KRB.
After washing, the
islets on nylon filters were placed in small glass vials. Added gently
to the vial were 400 µl of KRB supplemented with 10 mM
LiCl to prevent IP degradation and the appropriate agonists as
indicated. The vials were capped and gently gassed for 5 s with
95% O2, 5% CO2. After 30 min the generation
of IPs was stopped by adding 400 µl of 20% perchloric acid. Total
IPs formed were then measured using Dowex columns as described
previously (21, 22).
Hanks' solution was used for the islet isolation.
The perifusion medium consisted of 115 mM NaCl, 5 mM KCl, 2.2 mM CaCl2, 1 mM MgCl2, 24 mM NaHCO3,
and 0.17 g/dl bovine serum albumin. Other compounds were added as
indicated, and the solution was gassed with a mixture of 95%
O2, 5% CO2. The 125I-labeled
insulin used for the insulin assay was purchased from DuPont NEN. The
myo-[2-3H]inositol was purchased from Amersham
Corp. Bovine serum albumin (radioimmunoassay grade), glucose,
mannoheptulose, diazoxide, the sodium salt of pyruvate, and pyruvic
acid methyl ester as well as the salts used to make the Hanks'
solution and perifusion medium were purchased from
Sigma. Forskolin was purchased from Calbiochem.
Nitrendipine was a gift from Dr. A. Scriabine (Miles Institute for
Preclinical Pharmacology, West Haven, CT). Rat insulin standard (lot
615-ZS-157) was the generous gift of Dr. Gerald Gold (Lilly).
Collagenase (type P) was obtained from Boehringer Mannheim
Biochemicals.
Statistical significance was determined using
Student's t test for unpaired data or analysis of variance
in conjunction with the Newman-Keuls test for unpaired data. A
p < 0.05 was taken as significant. Values presented in
the figures and under "Results" represent the mean ± S.E. of
at least three observations.
RESULTS In the initial
series of experiments the dose dependence and the kinetics of the
insulin-secretory responses to MP were determined. In these studies
islets were perifused for 30 min in the absence of any added metabolic
fuel and were perifused for an additional 40 min with varying
concentrations of MP. As shown in Fig. 1, left, 5 mM MP had no insulinotropic effect,
whereas 10 mM stimulated a pronounced first phase of
release but no sustained insulin-secretory response. At a concentration
of 20 mM, MP evoked a large first phase of insulin
secretion and a sustained second-phase response (Fig. 1,
right). A higher level (40 mM) of MP reduced the
sustained release of the hormone when compared with 20 mM
MP (Fig. 1, right).
When compared to islets stimulated with
20 mM glucose (653 ± 198 pg/islet/min), peak
first-phase insulin-secretory responses to 20 mM MP
(728 ± 241 pg/islet/min) were comparable, but the second-phase
insulin-secretory responses to 20 mM glucose measured 35-40 min after the onset of stimulation (1592 ± 92 pg/islet/min) were about 6 times greater than those observed with 20 mM MP (253 ± 71 pg/islet/min; see Fig.
2). At a level of 20 mM and in agreement with previous results obtained with freshly studied rat islets (16) or cultured mouse islets (17), the sodium salt of pyruvate at
a level of 20 mM has no insulinotropic action (Fig. 2).
Mannoheptulose
(20 mM), which blocks glucokinase and abolishes
glucose-induced insulin secretion (4, 23, 24), had no inhibitory effect
on insulin release in response to 20 mM MP (Fig. 3, middle panel). Similar to its potentiating
effect on glucose-induced insulin secretion (25), the insulinotropic
effects of MP (20 mM) on the beta cell were amplified by
the addition of 10 µM forskolin (Fig. 3, lower
panel). Peak first-phase secretion to 20 mM MP in the
additional presence of forskolin averaged 1048 ± 186 pg/islet/min, whereas release rates measured 35-40 min after the onset
of stimulation increased to 436 ± 91 pg/islet/min. The
combination of 10 µM forskolin + 20 mM of the
sodium salt of pyruvate exerted no insulinotropic effect on the beta
cell (results not shown).
The stimulatory effect of 20 mM MP on insulin secretion was
blocked by 500 nM of the calcium channel antagonist
nitrendipine or by diazoxide (200 µM), which prevents
closure of the ATP-dependent potassium channel (26). In the
presence of nitrendipine or diazoxide, which were present during the
entire 70-min perifusion period, peak first-phase secretion was reduced
from 728 ± 241 pg/islet/min (control response in the absence of
inhibitor) to 93 ± 17 pg/islet/min (in the presence of
nitrendipine) or 107 ± 28 pg/islet/min (in the presence of
diazoxide). When compared with sustained release measured 35-40 min
after the onset of stimulation with MP alone (253 ± 71 pg/islet/min), release rates measured in the additional presence of
nitrendipine (17 ± 7 pg/islet/min) or diazoxide (17 ± 4 pg/islet/min) were also decreased.
In the next
series of experiments we examined the effects of MP on the PLC-mediated
hydrolysis of islet phosphoinositide (PI) pools. In the absence of any
added agonist, basal IP levels averaged approximately 2700 cpm/40
islets/30 min (Table I, line 1). Basal levels were not
significantly influenced by the addition of either nitrendipine or
diazoxide (Table I, lines 2 and 3). As shown in Table I, the addition
of MP to [3H]inositol-prelabeled islets resulted in a
significant increase in labeled IP accumulation, a sensitive index of
PLC activation (27-30). This response was dose-dependent,
with the maximal effect observed at 20 mM (Table I).
Significant effects on IP accumulation were evident with low levels
(2.5-5.0 mM) of MP (Table I, lines 4 and 5) devoid of any
sustained insulinotropic action (see Fig. 1). Like insulin secretion,
20 mM MP-induced IP accumulation was inhibitable by
diazoxide or nitrendipine. However, whereas insulin release was
abolished by either inhibitor, IP accumulation, although reduced, was
still significantly elevated above control levels (Table I, compare
lines 2 and 3 with lines 7 and 8). Mannoheptulose (20 mM)
had no deleterious effect on the capacity of 20 mM MP to
increase IP accumulation (Table I, line 9). The maximal stimulatory effect of 20 mM MP on PLC-mediated PI hydrolysis was
significantly less than that seen with 20 mM glucose (Table
I, compare lines 6 and 11). Consistent with its the lack of any
insulinotropic action, 20 mM of the sodium salt of pyruvate
had no effect on IP accumulation under these conditions (Table I, line
12).
Effects of various agonists on IP accumulation in isolated islets
Volume 272, Number 6,
Issue of February 7, 1997
pp. 3527-3531
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
EFFECTS ON INSULIN SECRETION, PHOSPHOINOSITIDE HYDROLYSIS,
AND SENSITIZATION OF THE BETA CELL*
and
-ketoisocaproate (6-10). Because these latter stimulants for
insulin secretion share in common many of the same stimulatory features
of glucose on the beta cell, the concept that mitochondrial-derived
signals mediate, at least in part, the stimulatory actions of the
hexose has been proposed (11, 12). For example, both glucose and
-ketoisocaproate stimulate similar changes in insulin secretion and
the calcium-dependent hydrolysis of islet phosphoinositide
(PI)1 pools and sensitize the beta cells to
subsequent restimulation, a phenomenon also referred to as priming or
time-dependent potentiation (TDP) (13-15).
Fig. 1.
Dose-dependent effects of MP on
insulin release from perifused islets. Groups of 14-18 freshly
isolated islets were perifused for 30 min in the absence of any
exogenously added fuel and perifused for an additional 40 min
(indicated by the vertical line in the figure) with 5.0 mM MP (left panel, 
), 10 mM MP
(left panel, 
), 20 mM MP (right
panel, ), or 40 mM MP (right panel, ). Mean values ± S.E. of at least three experiments are given. This and subsequent figures have not been corrected for the dead space
in the perifusion apparatus, 2.5 ml or 2.5 min with a flow rate of 1 ml/min.
[View Larger Version of this Image (18K GIF file)]
Fig. 2.
Comparison of the insulinotropic actions of
20 mM methyl pyruvate, 20 mM glucose, and 20 mM sodium pyruvate. Groups of 14-18 freshly isolated
islets were perifused for 30 min in the absence of any exogenous fuel
and perifused for an additional 40 min (indicated by the vertical
line in the figure) with 20 mM methyl pyruvate (),
20 mM glucose (), or 20 mM of the sodium salt of pyruvate (
). At least three experiments were performed under
each condition.
[View Larger Version of this Image (25K GIF file)]
Fig. 3.
Effect of mannoheptulose or forskolin on 20 mM MP-induced insulin secretion. Groups of 14-18
freshly isolated islets were perifused for 30 min in the absence of any
exogenous fuel and perifused for an additional 40 min (indicated by the
vertical line in the figure) with 20 mM MP alone
(, top panel), 20 mM MP + 20 mM
mannoheptulose (, middle panel), or 20 mM MP + 10 µM forskolin (
, bottom panel). At
least three experiments were performed under each condition.
[View Larger Version of this Image (25K GIF file)]
Stimulation condition
IP accumulation (cpm/40 islets/30 min)
1. No added agonist
2,694 ± 331
2. No added
agonist + nitrendipine
2,241 ± 240
3. No added agonist + diazoxide
2,728 ± 582
4. 5 mM MP
5,194
± 618
5. 10 mM MP
6,392 ± 267
6. 20 mM MP
8,255 ± 842
7. 20 mM MP + nitrendipine
5,295 ± 787
8. 20 mM MP + diazoxide
5,102 ± 494
9. 20 mM MP + MH
10,064 ± 2,388
10. 40 mM MP
6,319
± 598
11. 20 mM glucose
14,550 ± 1,783
12.
20 mM pyruvate (sodium salt)
2,276 ± 170
13. 30 mM KCl
5,614 ± 877
14. 30 mM KCl + nitrendipine
2,412 ± 269
We considered the possibility that the stimulatory effects of MP on IP accumulation were solely dependent on the capacity of MP to increase Ca2+ influx into the beta cell. To address this issue additional studies were conducted with depolarizing levels (30 mM) of potassium and the calcium antagonist nitrendipine (Table I, lines 13 and 14). From a quantitative perspective the magnitude of the effect of 20 mM MP on IP accumulation could not be duplicated by potassium-induced beta cell depolarization (Table I, compare lines 6 and 13). Most importantly, nitrendipine completely abolished potassium-induced IP accumulation but only partially reduced the impact of 30 mM potassium (Table I, compare lines 7 and 14).
MP Induces Time-dependent Potentiation of Insulin ReleaseMany structurally diverse molecules sensitize the beta
cell to subsequent restimulation. A common characteristic that links these agonists is their ability to increase PLC-mediated PI hydrolysis or to activate directly the enzyme protein kinase C. Because MP increases the activation of PLC, in the final set of experiments we
determined whether MP induces time-dependent potentiation
of insulin release. Similar to the effects of many compounds that increase PI hydrolysis in the beta cell, prior exposure to and subsequent removal of 20 mM MP primed or sensitized the
islet to subsequent 15 mM glucose stimulation. As shown in
Fig. 4, stimulation of control islets with 15 mM glucose after 60 min in 3 mM glucose resulted in a peak first-phase insulin response of 169 ± 36 pg/islet/min and a dramatic rising second phase of secretion. From a
quantitative perspective, when compared with prestimulatory secretion
rates of 28 ± 4 pg/islet/min, release rates measured 25-30 min
after the onset of 15 mM glucose stimulation increased
about 35-fold to 1102 ± 103 pg/islet/min. Prior exposure to 20 mM MP significantly amplified the first phase of release.
For example, peak first-phase release averaged 169 ± 36 pg/islet/min from naive islets, but this response increased to 734 ± 188 pg/islet/min from MP-pretreated islets. Second-phase release
rates measured 25-30 min after the onset of 15 mM glucose
stimulation were comparable in naive and MP-primed islets. Consistent
with the inability of the sodium salt of pyruvate to activate PLC and
with the concept that PLC activation plays an important role in the
induction of time-dependent potentiation, the sodium salt
of pyruvate did not induce TDP. Peak first-phase release rates to 15 mM glucose stimulation after exposure of islets to 20 mM pyruvate for 15 min averaged 160 ± 12 pg/islet/min
(results not shown). This response was not different from the response
of naive, unprimed islets to 15 mM glucose.
) was perifused for 60 min with 3 mM glucose followed
by a 30-min stimulatory period with 15 mM glucose. The
second group of islets () was perifused for 30 min with 3 mM glucose followed by 15 min with 3 mM glucose + 20 mM MP. After a 15-min washout in the presence of 3 mM glucose alone, these islets were restimulated with 15 mM glucose, and this is the period shown here. At least
five experiments were conducted under each condition.
DISCUSSION
Nutrient-stimulated insulin secretion depends on the metabolic transformation of fuel and the generation of appropriate intracellular signals. Recent studies have supported an important role for the ATP level or the ADP/ATP ratio as being an indispensable component of the signaling apparatus (31). In this view, increasing levels of ATP, a result of substrate intermediary metabolism via well-defined pathways, result in the closure of ATP-dependent K1+ channels and membrane depolarization. This event is quickly followed by opening of voltage-regulated Ca2+ channels. At least in freshly isolated rat islets, the increase in intracellular Ca2+ activates, along with yet to be defined mitochondrial signals, beta cell PLC (14, 32). This enzyme hydrolyzes islet PI pools, a process that generates inositol phosphates and stoichiometric amounts of diacylglycerol. Diacylglycerol and Ca2+ activate protein kinase C (33-35). The findings reported here with MP are compatible with these concepts of beta cell activation, and further comment on them is appropriate.
Methylation of potential metabolic substrates renders them more
effective insulin secretagogues. For example, whereas succinate alone
is an ineffective agonist for secretion, the methylated derivative of
succinate, monomethylsuccinate, is effective in this regard (7, 10).
Monomethylsuccinate not only stimulates insulin secretion in a
glucose-independent fashion, but prior exposure to it sensitizes the
islet to subsequent stimulation with glucose (18). In the present
studies and in agreement with a previous report using cultured mouse
islets (17), MP but not pyruvate evoked a significant insulin-secretory
response from freshly isolated rat islets. Like glucose-induced insulin
secretion, the stimulatory effects of MP were reduced by diazoxide and
nitrendipine. In this regard, MP is similar not only to glucose but to
glyceraldehyde, monomethylsuccinate, and -ketoisocaproate, and the
assumption that it acts via a pathway(s) that converges upon a common
metabolic pathway or generates a common signal seems reasonable. Thus,
MP via its metabolism generates ATP, which closes
ATP-dependent K1+ channels. Membrane
depolarization results in the subsequent opening of voltage-regulated
Ca2+ channels, influx of the divalent cation, activation of
PLC, and insulin secretion. The susceptibility of the insulinotropic
actions of MP to diazoxide and nitrendipine and the immunity of MP to mannoheptulose are consistent with this proposed mechanism of action.
At a higher level (40 mM), MP addition reduced the
secretory response of the islet. The basis for this inhibitory effect
was not explored further, but the results suggest caution when exposing islets to high levels of MP.
Similar to the actions of other nutrient molecules, MP activated beta cell PLC in a dose-dependent manner. The stimulatory effect of MP on IP accumulation was, like insulin release, immune to inhibition by mannoheptulose. Inositol phosphate accumulation in response to MP was reduced but not abolished by either diazoxide or nitrendipine. From a quantitative perspective the stimulatory effect of 20 mM MP on PLC activation was greater than that observed with depolarizing levels of potassium, an agonist whose stimulatory actions are dependent on calcium influx via voltage-regulated Ca2+ channels (26). These results suggest that more than just Ca2+ influx is involved the activation of PLC by nutrient agonists. Moreover, nitrendipine totally abolished the stimulatory action of potassium on IP accumulation, whereas it only partially reduced the effect of MP or glucose (36). These results lead to the conclusion that both calcium and a metabolic signal participate in the regulation of nutrient-activated, PLC-mediated PI hydrolysis and that these agonists (glucose and MP) but not high potassium alone are capable of generating both of these stimulatory signals.
We also performed a series of limited studies with the sodium salt of pyruvate. In agreement with previous reports (16, 17) it was without any insulinotropic effect on the beta cell even at a level of 20 mM. Inclusion of forskolin together with pyruvate, which theoretically at least should amplify even a weak pyruvate-generated stimulatory signal for secretion, was also without any positive effect. Furthermore, pyruvate had no effect on IP accumulation in islets.
Prior short-term exposure to 20 mM MP enhanced the
sensitivity of the islet to glucose stimulation. This amplified
response, referred to TDP or priming (37), is reproduced by a large
number of compounds that share in common the ability to activate one of
the several isozymes of PLC identified in islets (13, 15, 19, 38).
Thus, MP shares this property with such diverse agonists as carbachol,
acetylcholine, cholecystokinin, leucine, monomethylsuccinate, glucose,
-ketoisocaproate, and tolbutamide. As a working hypothesis we have
proposed that the induction of TDP is a result of an increase in
information flow in the PLC/protein kinase C signaling cascade (29, 30,
39). Because TDP is also produced by the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (40, 41), a unifying concept incorporates the activation of this enzyme and/or processes distal to it in its generation and maintenance. Not surprisingly, sodium pyruvate was impotent with regard to the induction of
time-dependent potentiation of insulin release.
From a quantitative perspective, these results differ from a previous study examining the insulinotropic effects of MP on the islet. For example, Mertz et al. (17) reported that 20 mM MP was a 6-fold greater stimulus for insulin secretion than was 12 mM glucose. With rat islets, although the first-phase secretory responses to 20 mM MP were comparable to 20 mM glucose, sustained second-phase release rates to MP were only 10-20% of those seen with 20 mM glucose. Several obvious points of departure exist between this and the previous report, however. We used freshly isolated rat islets, and insulin secretion was monitored during a dynamic perifusion. The report by Mertz et al. (17) used cultured mouse islets, and release was measured during static incubations. It is not known which of these or other methodological differences contribute to the quantitative disparity between the actions of MP and glucose on the beta cell. However, it is becoming increasingly apparent that the insulin-secretory responses and biochemical findings made with one rodent species may not necessarily be extrapolated to other rodent species or human beta cells (22, 42-47).
The precise nature of the metabolic signal or signals generated by MP that seem to interact with Ca2+ in the regulation of beta cell secretion and PLC activation is not known. For heuristic purposes at least, a common metabolic signal generated by all nutrient secretagogues seems to be a reasonable possibility. Based on our studies with nutrients metabolized exclusively within the mitochondria, we initially proposed that the signal was generated within this organelle. However, if the mitochondrial metabolism of glucose-derived pyruvate is exclusively responsible for the generation of this metabolic signal, then the results with sodium pyruvate remain problematic and difficult to interpret. This has to do with the inability of exogenously added pyruvate to activate either PLC or insulin secretion under conditions in which any small insulinotropic action of this compound should be manifest. A more global view might incorporate the concept that the metabolic signal may be generated by either cytosolic or mitochondrial pathways. For example, a recent report consistent with many other studies has suggested that ATP of either glycolytic or mitochondrial origin may be, along with Ca2+, the common signal involved in nutrient activation of the beta cell (31). In addition to regulating membrane polarization, ATP may serve as an important signaling molecule in other transduction systems as well, and the regulation of information flow in the PLC pathway may be one of them. This action of ATP may be exerted at multiple sites in the PLC/protein kinase C signaling cascade, including, but not confined to, the effect of ATP as a cofactor in protein kinase C phosphorylation events, the production of the variably phosphorylated phosphoinositides, or the transfer of phospholipids to the cell membrane (48). Other ATP-dependent steps have to be considered as well.
In conclusion, the findings presented demonstrate that the methylated and presumably more mitochondrial-permeable derivative of pyruvate stimulates the calcium-dependent activation of islet PLC and insulin release. Whereas the stimulatory actions of 20 mM MP on first-phase insulin secretion were comparable to 20 mM glucose, the effects of MP on sustained secretion were significantly less. MP induces TDP of release, a characteristic it shares in common with a variety of structurally distinct molecules linked by their ability to activate PLC directly or the enzyme protein kinase C (13, 15, 18, 19, 37, 38, 40, 41, 49). The judicious use of MP may prove to be a valuable tool in defining the nature of the biochemical factors and transduction pathways that interact so elegantly to regulate insulin secretion from the pancreatic beta cell.
FOOTNOTES
To whom correspondence should be addressed: Yale University School
of Nursing, 100 Church St. South, P. O. Box 9740, New Haven, CT
06536-0740. Tel.: 203-785-5522; Fax: 203-785-6455; E-mail: Walter.Zawalich@Yale.Edu.
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Y.-P. Zhou, J. C. Pena, M. W. Roe, A. Mittal, M. Levisetti, A. C. Baldwin, W. Pugh, D. Ostrega, N. Ahmed, V. P. Bindokas, et al. Overexpression of Bcl-xL in beta -cells prevents cell death but impairs mitochondrial signal for insulin secretion Am J Physiol Endocrinol Metab, February 1, 2000; 278(2): E340 - E351. [Abstract] [Full Text] [PDF]
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ABSTRACT