arachidonic acid metabolism in isolated pancreatic islets: ii. the effects of glucose and of...
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3iochjmicu et Biophysics A era, 794 (I 984) 125 - 136
Elsevier
125
BBA 51643
A~CHIDONIC ACID METABOLISM IN ISOLATED PANCREATIC ISLETS
Ii. THE EFFECTS OF GLUCOSE AND OF INHIBITORS OF ARACHIDONATE METABOLISM ON INSULIN SECRETION AND METABOLITE SYNTHESIS
JOHN TURK a.h.*. JERRY R. COLCA ‘, NIRMALA KOTAGAL h and MICHAEL L. MCDANIEL ’
* Division of Laboraro~ Medicine and Departments of Medicme, Pharmacology, and h Pathology, Washington Universitv School of Medicine, St. Louis, MO 63110 (U.S.A.)
(Received November 29th. 1983)
Kqy words: Arachidonic acid metabolism; Glucose; Insulin secretion; (Pancreatic islet)
Isolated pancreatic islets from the rat incubated with 28 mM glucose have been found to secrete more insulin and to synthesize greater mounts of arachidonate lipoxygenase and cyclooxygenase products than islets incubate with 3 mM glucose. This effect was not apparent in studies examining metabolism of radiola~led arachidonate and was revealed only when the metabolites were quantitated with mass spectrometric measurements. That the glucose-induced synthesis of arachidonate metabolites may participate in, insulin secretion was suggested by studies with inhibitors of arachidonate metabolism. Eicosa 5,8,11,14 tetrynoic acid (ETYA) suppressed glucose-induced insulin secretion by 63-74% at a concentration (20 pM) which inhibited the synthesis of arachidonate lipoxygenase and cyclooxygenase products by 90%. Indomethacin (10 pM) completely prevented islet synthesis of cyclooxygenase products but did not influence glucose-induced insulin secretion. Although indomethacin did not inhibit the conve~ion of exogenous, 3H-labeled ~achidonate to J3H]12-HETE, it did si~ifi~n~y inhibit (41-72%} the synthesis of 12-HETE from endogenous precursor. This is presumed to reflect indirect effects of indomethacin on hydrolysis of arachidonate from phospholi- pids, as recently reported in platelets. These studies constitute the first demonstration that glucose stimulates the synthesis of a lipoxygenase product (12-HETE) from endogenous arachidonate by isolated islets, and that suppression of 12-HETE synthesis with ETYA reduces glucose-induced insulin secretion from isolated islets.
Introduction
* To whom correspondence and reprint requests should be
addressed at: Box 8118, Washington University School of
Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110,
U.S.A.
Abbreviations: HETE, hydroxyeicosatetraenoic acid; ETYA,
eicosa-5,8,11,14-tet~noic acid; HPETE. hydroperoxyeicosa-
tetraenoic acid; GC, gas chromatography; MS, mass spectrom- etry: HPLC, high-performance liquid chromatography; RP.
reversed phase; NP, normal phase; EI, electron impact; CL
chemical ionization: NI, negative ion: HHT, 12-hydroxy-
5,8,10-heptadecatrienoic acid: Hepes, 4-(2-hydroxyethyI)-l-
piperazineethanesulfonic acid; ME, methyl ester; PFBE, penta-
fluorobenzyt ester; MO, methoxime; TMS, trimethylsilyl.
interest in the possibility that metabolites of arachidonic acid participate in the regulation of
insulin secretion has been generated by several studies with inhibitors of arachidonate metabo- lism. Arachidonate cyclooxygenase inhibitors have
been reported to augment glucose-induced insulin secretion in human subjects [l-4] and in cultured pancreatic cells from rat neonates [S], although others have reported that cyclooxygenase inhibi- tors suppress glucose-induced insulin release in human subjects [6] or do not influence insulin
~5-276O/S4/$03.~ 0 1984 Elsevier Science Publishers B.V.
126
release from perfused pancreas preparations [7] or isolated islets [8].
Discrepancies between these studies may relate
in part to the differing natures of the systems
chosen for the study of insulin secretion. Infusion
of cyclooxygenase inhibitors into intact organisms
likely suppresses prostaglandin biosynthesis in ex-
tra-pancreatic tissues as well as in islets and this may result in hormonal, metabolic or hemody-
namic effects independent of islet prostaglandin synthesis which influence insulin secretion. The
effects of inhibitors of arachidonate metabolism
on glucose-induced insulin secretion from cultured
neonatal rat pancreatic cells are difficult to inter-
pret, since these preparations exhibit a weak [9] or
absent [5] insulin secretory response to glucose and
therefore presumably do not retain a normal
stimulus-secretion mechanism. Intact isolated islets
exhibit a robust insulin secretory response to glu-
cose [lO,ll] and are probably the most appropriate
preparation with which to study the influence of
arachidonate metabolism on glucose-induced in-
sulin secretion. There is, however, little informa-
tion on the effects of inhibitors of arachidonate metabolism on concomitantly quantitated insulin
secretion and metabolite biosynthesis by isolated
islets. Cyclooxygenase inhibitors exert effects in addi-
tion to suppression of prostaglandin biosynthesis
[12-l 91, including enhancement of the metabolism
of free arachidonate via lipoxygenase pathways
[20]. Inhibitors of arachidonate lipoxygenases have
been reported to suppress insulin secretion
[9,21-241, suggesting that lipoxygenase products
may participate in insulin secretion. Lipoxygenase
inhibitors also have multiple actions, however, in-
cluding interference with arachidonate oxygena-
tion by the cyclooxygenase [20] and possibly by
monoxygenases of the cytochrome P-450 type
[25,26]. One requirement for interpreting the effects of
these inhibitors on insulin secretion would be the determination of the actual influence of these com- pounds on islet synthesis of metabolites from en- dogenous arachidonate at concentrations of the inhibitors which influence insulin secretion. Fur- ther evaluation of the possibility that arachidonate metabolites participate in glucose-induced insulin secretion would also require examination of the
influence of glucose on islet synthesis of these
metabolites. Neither of these issues has been ade-
quately addressed by previous studies of
arachidonate metabolism by isolated islets, due in
part to the technical difficulties of quantitating
arachidonate lipoxygenase products in the amounts
synthesized by islets from endogenous precursor.
We have obtained information on these issues
by quantitating islet synthesis of both arachidonate
lipoxygenase and cyclooxygenase products by
sequential HPLC analyses followed by capillary column @Z-negative-ion chemical ionization mass
spectrometric measurements with deuterated inter-
nal standards. The effects of glucose and of inhibi-
tors of arachidonate metabolism have been de-
termined on concomitantly measured insulin secre-
tion and metabolite synthesis by isolated islets.
Materials and Methods
All materials were obtained from the sources indicated in the accompanying paper [29]. The
procedures for metabolite recovery, analysis, de- rivatization and quantitation are also described in
that paper [29], as are the procedures for islet
isolation and culture. For studies examining the influence of glucose
on arachidonate metabolism and on insulin secre-
tion, isolated islets (approx. 1.5 ’ 104) were pre-
incubated (30 min, 37’C) in medium (5 mM Hepes,
135 mM NaCl, 24 mM NaHCO,, 5 mM KCl, 1 mM MgCl, and 2.5 mM CaCl,, pH 7.4) supple-
mented with glucose (3 mM) and bovine serum albumin (0.1%). The islets were then collected by centrifugation, resuspended in fresh medium of
the same composition, and divided into 2-8 groups in individual siliconized vials. Incubations were
initiated by addition of glucose (final concentra-
tion 3 mM or 28 mM) and continued for 20 min at
37°C. At the end of this period an aliquot of medium was withdrawn for radioimmunoassay of secreted insulin [30], and a mixture of suitably labeled internal standards of arachidonate metabolites was added in methanol (final con- centration 15%) as described [29]. Particulate
matter was removed by centrifugation (3000 x g, 5 min). Insulin from the protein pellet was extracted with 75% ethanol/l.5% HCl and measured by radioimmunoassay, as was the insulin in an aliquot
127
of the methanolic supernatant. This provided a
measure of the total insulin content of each group
of islets and indicated that the islet mass varied by
less than 7% between groups. The remainder of the
methanolic supernatant was analyzed for content
of arachidonate metabolites as described [29].
For studies examining the effects of ETYA and
indomethacin on arachidonate metabolism, a pro-
cedure similar to that described above was fol- lowed except that: (a) ETYA (20 or 50 PM) or
indomethacin (5 or 10 PM) was included in the
preincubation and incubation media for islets
treated with these inhibitors, and (b) albumin was
excluded from both preincubation and incubation
media. (Albumin was found to prevent any effect
of the inhibitors, presumably due to avid drug-
protein binding.) Insulin secretion was difficult to
measure reliably in albumin-free medium. For ex-
periments examining the influence of ETYA and
indomethacin on insulin secretion the following
protocol was therefore employed. 20 islets were
randomly selected under a stereomicroscope for
each sample. The islets were incubated (20 min, 37°C) in albumin-free medium (200 ~1) containing
glucose (3 or 28 mM) and ETYA (20 PM) or
indomethacin (10 PM). At the end of the incuba-
tion period an equal volume of medium containing
bovine serum albumin (0.2%) was added to aid in
the recovery of secreted insulin. An aliquot was then withdrawn for the radioimmunoassay of in-
sulin.
Results
The question of whether a stimulus to insulin secretion (glucose) would influence islet biosynthe-
sis of arachidonate metabolites was first addressed
with islets prelabeled with [ 3H,]arachidonate and then washed free of unincorporated radiolabel by
repeated low-speed centrifugation and resuspen-
sion in fresh medium. The prelabeled islets were
then incubated with a low (5 mM) or high (28
mM) concentration of glucose for 20 min at 37°C
in the absence of additional radiolabeled substrate, and the incubations were terminated by the addi- tion of a methanolic solution of a mixture of 14C-labeled internal standards as described [29]. The products were then extracted and analyzed by RP-HPLC. The ratio of 3H to 14C label in the
various peaks (e.g., 12-HETE) provided an index
of the conversion of incorporated [ 3H]arachidonate
to products. Islets prelabeled in this way generated a profile
of products on RP-HPLC similar to unlabeled
islets incubated directly with free [ 3H]arachidon-
ate, although the relative abundance of the prod-
ucts differed (panels A and B, Fig. 1). Comparison
of the product profile from islets which were pre-
labeled and then incubated with 5 or 28 mM
glucose revealed no apparent difference in the ‘H
to 14C ratios in the 12-HETE peaks (panels B and
C Fig. 1). No difference in this ratio was apparent
even when the 12-HETE peaks were extracted
from the RP-HPLC solvent, converted to the methyl esters, and analyzed by NP-HPLC (column
II, Solvent K) (not shown). Further analyses of the cyclooxygenase products (column I, solvent A then
column II, solvent I) also failed to reveal dif-
ferences in the 3H-to-‘4C ratios for these com-
pounds under the two experimental conditions
(not shown). Ionophore A23187 also did not
markedly influence the 3H-to’4C ratio in the 12-
HETE peak (panel D, Fig. l), although the prod-
uct-profile from A23187-treated cells exhibited
smaller amounts of ‘H-label in two unidentified
peaks eluting just before and just after the HETE
species. Similar experiments comparing 3 mM
(rather than 5 mM) to 28 mM glucose also failed
to demonstrate any apparent influence of the glu-
cose concentration on the conversion of incorpo-
rated [‘Hlarachidonate to [3H]12-HETE or cyclooxygenase products (not shown).
It was considered possible that several pools of
arachidonate might exist within the islet cells and
that exogenous [3H]arachidonate might not be in- corporated into all pools with the same specific
activity. If glucose recognition were coupled to a
pool of arachidonate with a low specific activity,
the generation of [3H]12-HETE might not reflect
total synthesis of 12-HETE in response to a glu-
cose challenge.
To address this possibility, 12-HETE was quan-
tified by a stable isotope dilution GC-MS assay using octadeutero-1ZHETE as internal standard as described in the preceding manuscript [29]. Similar measurements of prostaglandin E, and prostaglandin F,, were performed with deuterated internal standards. Freshly isolated islets in-
A) SuPCrnotont from Pre-iobell!ng Islets Wath
3H-Ar-ochldonate
TxB2. PGE, LTE& HHT 12 H;TE 5 HETE TxB,. PGE, LTB4 HHT 12 HETE 5HETE
B)Control lncubatlon of 3H-Labelled Islets
(5mM Glucose I
6
TxB2. PGE LT$ HHT 2
12 HETE 5HETE
C) 3H-LobelIed Islets and 28m~ GIUCOS~
SOLVENT F SOLVENT G
6
%
?
1.0 i ‘: 0
N 40
; ; a a ” ”
I Q5 ” F)
29
DJ3H-Labelled Islets and A23187
SOLVENT F SOLVENT G
Tn0>+ PGEp LTB4 HHT 12HETE SHETE
ELUTION VOLUME (ML)
Fig. 1. Influence of glucose concentration on synthesis of ‘H-labeled metabolites by islets prelabeled with [ ‘Hlarachidonate. Islets
(approx. 1.5.104) were isolated from 30 rats as described [29] and incubated with [‘Hlarachidonic acid (100 PCi) for 1 h at 37’C in
albumin-free medium. The islets were then repeatedly washed by low-speed centrifugation and resuspension in fresh incubation medium which contained no additional radiolabel. The ‘H-labeled islets were then split into three equal populations and incubated
with 5 mM glucose (panel B), 28 mM glucose (panel C). or 5 mM glucose and 10 pM A23187 (panel D). Incubations were continued
for 20 min at 37°C and were terminated by the addition of a methanolic solution of 14C-labeled standard arachidonate metabolites as
described [29]. The products were extracted and subjected to RP-HPLC (column 1, solvent F. then solvent G). The chromatograms are
shown in the figure. Panel A is the chromatogram of products obtained from the supernatant of the initial labeling incubation and
presumably reflects metabolism of free 13H]arachidonate during the labeling period. Panels B, C and D presumably reflect
metabolism of [ ‘Hlarachidonate which had initially been esterified into membrane lipid and which was subsequently released during
incubation under the various experimental conditions. No striking difference was observed between panels B, C and D in the
“C-to’H ratios for any of the known metabolites. This impression was confirmed by collecting each 14C-peak separately and
subjecting it to NP-HPLC analysis. A similar experiment was performed comparing the effects of 3 mM and 28 mM glucose. and similar results were obtained. The 3H-labeled material eluting just after [14C]5-HETE was initially thought to be the delta-lactone of
5-HETE. This was shown not to be the case, since the material was inert to treatment under alkaline conditions which result in
hydrolysis of such lactones [31]. Such behavior would be expected for a non-allylic epoxide of arachidonate [31].
cubated with 28 mM glucose for 20 min secreted
more insulin and synthesized greater amounts of 12HETE than did islets incubated with 3 mM
glucose (Table I). This effect was significant at the
P < 0.005 Ievel.
A similar experiment was performed with islets
which had been cultured overnight. Intact islets
cultured in this way synthesize greater amounts of
arachidonate metabolites than freshly isolated islets [29]. These cultured islet preparations were used in
the preceding manuscript [29] to exclude en- trapped platelets as the source of islet-derived arachidonate metabolites. The synthesis of
arachidonate metabolites by cuhured islets in
medium containing 3 or 28 mM glucose is sum-
marized in Table II. Cultured islets incubated with
28 mM glucose secreted more insulin and synthe-
sized larger amounts of 1ZHETE than did islets
incubated with 3 mM glucose. This effect was
significant at the P < 0.02 level. Glucose (28 mM)
also appeared to stimulate synthesis of each of the
three cyclooxygenase products measured under these conditions, but there was considerable varia-
TABLE 1
129
tion between experiments in the absolute amounts
of the cyclooxygenase products synthesized by the islets. The ratio of the amount of cyclooxygenase
product synthesized at 28 mM glucose to that synthesized at 3 mM glucose was significantIy
greater than one for prostaglandin E,, pros-
taglandin F,, and thromboxane B, at the P -c 0.05
level (Table II) when the ratios were calculated
separately for each independent experiment.
The observation that glucose increased the
synthesis of arachidonate metabolites raised the
question of whether the glucose-induced synthesis
of arachidonate metabolites played a role in in-
sulin secretion or was merely an epiphenomenon
associated with the cellular response to glucose but
not involved in stimulus-secretion coupling. This
question was addressed by examining the effects
of inhibitors of arachidonate metabolism on glu-
cose-induced insulin secretion. The non-selective
lipoxygenase and cyclooxygenase in~bitor eicosa- 5,8,11,14-tetrynoic acid (ETYA) (20 (*M) (201 did
not suppress basal insulin secretion at 3 mM glu- cose by islets cultured overnight (Table III). ETYA
INFLUENCE OF GLUCOSE CONCENTRATION ON PRODUCTION OF METABOLITES FROM ENDOGENOUS
ARACHIDONATE BY FRESHLY ISOLATED PANCREATIC ISLETS
For each experiment pancreatic islets (approx. 1.5~10’) were isolated from 30 rats as described 1291, incubated for 30 min at 37°C in
KRB medium containing glucose (3 mM) and bovine serum albumin (0.1%). collected by centrifugation and resuspended in fresh
medium. The islets were then divided into eight equal populations and incubated for 20 min at 37°C in KRB medium containing
glucose (3 or 28 mM) and bovine serum albumin (0.1%). At the end of the incubation period an aliquot of the medium was withdrawn
for the radioimmunoassay of insulin and a methanolic solution of a mixture of *H- and 3H-labeled internal standards was added. The
products were extracted, isolated by HPLC, and quantitated by GC-MS (NCI) as described [29]. Equality of islet mass in each
population was confirmed by measurement of acid-ethanol extractable insulin as described in the methods section. The tabulated
amounts are the means of eight independent determinations on separate populations of islets. Standard errors of the mean are
indicated. The ratio was calculated as the amount of metabolite measured in a given sample divided by the mean amount of
metabolite measured at 3 mM glucose in the same experiment. The P values for differences between Entries 1 and 2 were calculated
with Student’s f test.
Entry [glucose] Metabolite
(mM) Prostaglandin E, Prostaglandin Fao 12-HETE Insulin secretion
Amount Ratio Amount Ratio Amount Ratio Amount Ratio
(PP) (Pg) (PP) (mu)
1 3 43 1.00 79 1.00 1550 1 .oo 8.1 1.00 +14 kO.13 *15 $- 0.04 +210 + 0.04 f 3.4 f 0.06
2 28 89 2.40 120 1.35 2660 1.58 66.6 9.45 +21 + 0.67 +38 kO.15 4 200 +0.14 + 6.4 * 1.5
P value for P < 0.10 P i 0.10 P < 0.50 P -c 0.05 P < 0.002 P -c 0.005 P e 0.001 P < 0.001 difference between P > 0.05 P > 0.05 P z 0.20 P > 0.02 P > 0.001 P > 0.002 P > 0 PBO entries 1 and 2
I30
TABLE II
INFLUENCE OF GLUCOSE CONCENTRATION ON PRODUCTION OF METABOLITES FROM ENDOGENOUS
ARACHIDONATE BY ISOLATED PANCREATIC ISLETS THAT HAD BEEN CULTURED OVERNIGHT
Experimental conditions were identical to those in Table I except that all islets were cultured overnight before study: bovine serum
albumin was excluded from the incubation medium; and n = 4. A similar experiment was performed in the presence of bovine serum
albumin (n = 2). and similar results were obtained: at 3 mM glucose the mean amounts (pg) of metabolites produced were:
prostaglandin E, (461+3), prostaglandin F,, (75$:0), thromboxane Bz (157+45). IZ-HETE (#OS& 336). At 28 mM glucose the
mean amounts (pg) of metabolites produced were: prostaglandin E, (695 k 116). prostagiandin F,, (136 5 13). thromboxane B,
(167+86). I2-HETE (6957+ 1863). Under these conditions insulin secretion at 3 mM glucose was 14.8kO.7 mU and at 28 mM
glucose was 70.8 + 0.1 mu.
Entry [GLucoseI Metabolite
(mM) Prostaglandin E, Prostagiandin F2n Thromboxane B, 12-HETE
Amount Ratio Amount Ratio Amount Ratio Amount Ratio
(pg) (PP) (Pg) (mu)
1 3 1972 1 .oo 220 1. .oo 286 1.00 2800 1 .oo
&855 kO.17 +38 * 0.07 F73 * 0.08 + 623 & 0.24
2 28 3017 1.83 393 1.90 613 2.33 7540 2.17
+ 853 i 0.24 +17 rl: 0.29 +195 + 0.29 + 1000 i 0.50
P value for P < 0.50 P -c 0.05 P < 0.01 P i 0.05 P i 0.20 P < 0.005 P < 0.01 P < 0.02 difference between P z 0.20 P > 0.02 P > 0.005 P 3 0.02 P > 0.10 P > 0.002 P > 0.005 P > 0.01 Entries 1 and 2
did reduce the amount of insulin secreted from cultured islets in response to 28 mM glucose by
about 63% (Table III). ETYA also inhibited glu-
cose-induced insulin secretion from freshly iso-
iated islets (Table IV). This suggested that the
glucose-induced synthesis of IZHETE or of a
cyclooxygenase product participated in the insulin
secretory response to glucose. Experiments with
indomethacin suggested that the cyclooxygenase
products were not involved in this response. In- domethacin (5 or 10 PM) did not significantly
influence the amount of insulin secreted by cul-
tured islets incubated with 28 mM glucose (Table
III). Indomethacin also did not influence glucose-
induced insulin secretion by freshly isolated islets (not shown).
To determine whether these inhibitors had the expected influence on islet synthesis of metabolites from endogenous arachidonate, synthesis of the metabolites was quantitated in the presence and absence of the inhibitors. ETYA at a concentra- tion (20 PM) which suppressed glucose-induced insulin secretion also suppressed the synthesis of both lipoxygenase and cyclooxygenase products by
about 90% both at 3 and 28 mM glucose (Table
V). Indomethacin completely suppressed the
synthesis of cyclooxygenase products (Table V) at
a concentration (10 FM) which did not influence
glucose-induced insulin secretion. A similar degree of cyclooxygenase inhibition by indomethacin (5
or 10 PM) was observed at 3 mM and at 28 mM
glucose (Table V). Unexpectedly, indomethacin (5 or 10 FM) also
suppressed the synthesis of the lipoxygenase prod-
uct IZI-IETE from endogenous arachidonate by 61 i 7% (Table V). This effect was observed both with 3 mM and 28 mM glucose. It is unlikely that
this reflects direct inhibition of the islet 12-lipo-
xygenase by indomethacin, since the conversion of exogenous 3H-labeled arachidonate to [ ‘H]12- HETE by isolated islets was not inhibited by indomethacin 1291. It is possible that suppression of 12-HETE synthesis from endogenous arachidonate by indomethacin reflects the pres- ence in islets of phospholipase(s) requiring the generation of cyclooxygenase products for maxi- mal activation. This phenomenon has been de- scribed in platelets by two independent groups of
131
TABLE III
INFLUENCE OF INHIBITORS OF ARACHIDONATE METABOLISM ON INSULIN SECRETION BY ISOLATED PAN-
CREATIC ISLETS THAT HAD BEEN CULTURED OVERNIGHT
Pancreatic islets were isolated from 10 rats on each of three separate occasions and cultured overnight as described 1291. Culture
medium was then removed after low-speed centrifugation and the islets were resuspended in fresh incubation medium. Twenty islets
were randomly selected for each sample under a stereo-microscope and three independent samples were prepared for each
experimental condition. The islets were preincubated for 30 min at 37°C in incubation medium containing 3 mM glucose with or
without an inhibitor. (Albumin was excluded from the medium, since it was found that albumin prevented any effect by the inhibitors,
presumably due to drug-protein binding.) The preincubation medium was then removed and replaced by medium containing glucose
(3 or 28 mM) with or without inhibitor. Incubations were continued for 20 min at 37°C. At the end of this interval, bovine serum
albumin (final concentration 0.1%) was added to facilitate recovery of secreted insulin, and aliquots of the incubation medium were
withdrawn for the radioimmunoassay of insulin. Tabulated secretion rates represent means of triplicate measurements on each of
three independent samples. Standard errors of the mean are indicated (n = 3)
Entry [Glucose]
(mM)
Inhibitor Insulin
secretion
(pU/min per islet)
1 3
2 28
3 3
4 28
5 3
6 28
7 3
8 28
9 3
10 28
11 3
12 28
Fraction of control increment with ETYA (20 PM)
(Ent~4-Ent~3)/(~t~2-Ent~ 1)
P value for ETYA effect (difference between
Entries 2 and 4)
None
None
ETYA
(20 BM) ETYA
(20 FM) ETYA
(50 PM) ETYA
(50 PM)
None
None
Indomethacin
(5 PM) Indomethacin
(5 idM) Indomethacin
(10 FM) lndomethacin
(10 PM)
0.40*0.11
8.75 f 0.25
1.04 f 0.08
4.14 f 0.39
0.69 i: 0.10
3.94kO.21
0.83 -I_ 0.40
8.19 ho.48
0.60~0.11
7.45 + 1.26
0.5750.12
8.59 f 0.21
0.37
0<P<0.001
Fraction of control increment with indomethacin (10 PM)
(Entry 12 - Entry ll)/(Entry 8 - Entry 7)
P value for indomethacin effect
(difference between Entries 8 and 12)
1.08
0.5 < P <: 1
investigators 132,331. Direct inhibition of di- acylglycerol lipase by indomethacin has also been reported [19], but much higher concentrations of indomethacin (50% inhibition at 200 PM) than those employed in this study are required for this effect.
Discussion
A number of studies involving exogenous metabolites and inhibitors have provided cir- cumstantial evidence that arachidonate metabo- Iism may be involved in insulin secretion. The
132
TABLE IV
INFLUENCE OF ETYA ON GLUCOSE-INDUCED INSULIN SECRETION BY FRESHLY ISOLATED PANCREATIC
ISLETS
Pancreatic islets were isolated from 10 rats on each of five occasions and studied on the day of harvest. Conditions were otherwise
identical to those described in Table III. Tabulated secretion rates represent means of triplicate measurements of each of five
independent samples. Standard errors of the mean are indicated (n = 5).
Entry [Glucose]
(mM)
Inhibitor Insulin
secretion
(pU/min per islet)
1 3 None 1.43 k 0.29
2 28 None 5.97*0.3s
3 3 ETYA 1.33t0.27
(20 PM) 4 28 ETYA 2.53 0.39 k
(20 PM)
Fraction of control increment with ETYA (20 PM)
(Entry4-Entry 3)/(Entry 2-Entry 1) 0.26
P value for ETYA effect
(difference between Entries 2 and 4)
0 <P < 0.001
TABLE V
INHIBITION BY INDOMETHACIN AND ETYA OF PRODUCTION OF METABOLITES FROM ENDOGENOUS
ARACHIDONATE BY ISOLATED PANCREATIC ISLETS
Experimental conditions were identical to those in Table II except that, as indicated, the incubations were performed in the presence
and absence of indomethacin (5 or 10 PM) or ETYA (20 pM). This table summarizes the results of three separate experiments in
which the quantities of the indicated metabolite were measured by gas chromatographic-mass spectrometric stable isotope dilution
methods as described 1291. The Percent inhibition of metabolite synthesis was calculated by dividing the quantity of a given metabolite
produced in the presence of inhibitor by that produced in the absence of inhibitor at the same glucose concentration in the same
experiment, multiplying the resulting ratio by 100, and subtracting the product from 100. In each experiment measurements were
performed on two independent samples at each condition tested.
[Glucose]
(mM)
3
Inhibitor
ETYA
(20 PM)
Inhibition of metabolite synthesis (%)
Prostaglandin E, Prostaglandin Fzo
94 88
Thromboxane B, I2-HETE
82 88
28
3
ETYA
(20 PM)
indomethacin
(5 PM)
92 85 90 88
100 99 96 71
28 indomethacin
(5 PM)
100 98 98 42
3
28
indomethacin
(IO ILM) indomethacin
(10 PM)
96 95 94 59
100 98 98 12
133
physiologic importance of these observations has
been uncertain due to the lack of direct informa- tion on the metabolism of endogenous
arachidonate by islets and on how stimuli to in-
sulin secretion affect islet arachidonate metabo- lism. We have examined the effects of an insulin
secretagogue and of inhibitors of arachidonic acid
metabolism both on insulin secretion and on the
metabolism of endogenous arachidonate by intact,
isolated pancreatic islets. Our observations indi-
cate that metabolites derived from endogenous
arachidonate may participate in the secretion of
insulin from isolated islets which is induced by
glucose.
The observations presented here and in the
preceding paper [29] indicate that the metabolism
of exogenous, radiolabeled arachidonate by iso-
lated pancreatic islets differs significantly from the metabolism of endogenous arachidonate. Such dis-
crepancies have also been described for platelets [34], with which islets appear to share many fea-
tures of arachidonate metabolism. Discrepancies between the metabolism of exogenous and endoge-
nous substrate were apparent from examination of the effects of glucose and of indomethacin on islet
synthesis of ~achidonate metabolites.
Experiments in which islets were prelabeled with
exogenous [ ‘Hjarachidonate and then exposed to different concentrations of glucose suggested that
little change in the metabolism of arachidonate
was induced by a concentration of glucose which
stimulated insulin secretion. When this experiment was repeated with unlabeled islets and the amounts
of various ~achidonate metabolites derived from
endogenous precursor were quantitated, it became
apparent that a glucose concentration which stimulated insulin secretion also stimulated the
synthesis of both arachidonate hpoxygenase and cyclooxygenase products. These observations sug-
gest that at least two pools of arachidonate exist within the islets, which can be distinguished by the
degree to which they incorporate exogenous arachidonate and by their relative contribution of
precursor to arachidonate 12-lipoxygenase and cyclooxygenase at substimulatory (3 mM) and stimulatory (28 mM) concentrations of glucose.
The fact that glucose stimulates the synthesis of arachidonate metabolites suggests that glucose may increase the availability of free arachidonate sub-
strate to the islet 12-lipoxygenase and cycloo-
xygenase. This could occur if glucose stimulated phospholipases, and glucose-induced changes in
arachidonate turnover in the phospholipids of islet membranes have been reported [35,36]. Phos-
pholipase inhibitors have variously been reported
to suppress (35,373, enhance [3g], or to exert com-
plex, time-dependent effects [36] on glucose-in- duced insulin release. These agents have multiple
effects in addition to phospholipase inhibition [39], however, and their influence on islet metabolism
of endogenous arachidonate is not known.
The magnitude of the stimulatory effect of glu-
cose on islet production of arachidonate metabo-
lites is small (about 2-fold) compared to the mag- nitude of the change in glucose concentration (3
vs. 28 mM) or to the change in insulin secretion
(5-&fold). This 2-fold increase is similar to that
observed for cyclic AMP content of isolated pan-
creatic islets from the rat exposed to 2 vs. 27 mM glucose for 20 min [40], however, and substantial
evidence suggests that cyclic AMP is an important
modulator of insulin secretion [40-421. In addi- tion, 2-fold increments in 12-HETE production
are associated with a significant biological re-
sponse in other processes in which arachidonate
lipoxygenase products are thought to participate,
such as eosinophil migration [43,44]. Another
group of investigators has recently reported that
glucose increases the synthesis of an arachidonate
cyclooxygenase product by isolated islets [46]. The
magnitude of the observed effect was similar to
that reported here. These investigators did not
examine the synthesis of lipoxygenase products
from endogenous arachidonate by islets.
That the synthesis of arachidonate metabolites induced by glucose may participate in the secre-
tion of insulin is suggested by effects of inhibitors of arachidonate metabolism on insulin secretion, ETYA suppressed glucose-induced insulin secre-
tion by 63% at a concentration (20 PM) which reduced islet synthesis of both 12-HETE and
cyclooxygenase products from endogenous arachidonate by 90%. Since indomethacin did not influence glucose-induced insulin secretion at a concentration (10 PM) which completely pre- vented islet synthesis of cyclooxygenase products, the inhibition of secretion by ETYA may reflect a role for IZHETE or its precursor IZHPETE in
134
the regulation of insulin secretion.
Surprisingly, indomethacin was also found to
reduce the amount of 12-HETE formed from en-
dogenous precursor but not from exogenous, ‘H-
labeled arachidonate [29]. These findings and other published experience with indomethacin [20] sug-
gest that the compound does not directly inhibit
the arachidonate 12-lipoxygenase. Indomethacin
may rather impair substrate availability to the
12-lipoxygenase. Two independent groups of in-
vestigators have recently reported that collagen-in-
duced arachidonate release from platelets is in-
hibited by indomethacin (and aspirin) and that this inhibition may be overcome with exogenous
prostaglandin H, or an analog of prostaglandin
H, [32,33]. Indomethacin also inhibited collagen-
induced hydrolysis of phosphatidylinositol to di-
acylglycerol [32] and formation of phosphatidic
acid [33] in these systems, and both of these effects could also be reversed with prostaglandin H, or an
analog of prostaglandin H, [32,33]. These findings
suggest that arachidonate cyclooxygenase products are required for maximal hydrolysis of phos-
phatidylinositol and arachidonate release in some circumstances. This requirement is not universal,
since thrombin-induced phosphatidylinositol hy-
drolysis and arachidonate release from platelets is unaffected by indomethacin [32,33].
The observations that indomethacin suppresses
12-HETE production by islets but does not in-
fluence glucose-induced insulin release suggest that
the reduction in glucose-induced insulin secretion
by ETYA may be due to some action of ETYA other than its inhibition of the arachidonate 12-
lipoxygenase. It is not possible to exclude a role
for 12-lipoxygenase products in modulating glu- cose-induced insulin release for several reasons,
however. First, the suppression of 12-HETE
synthesis by indomethacin was less complete than that by ETYA, and the residual synthesis of 12- lipoxygenase products may have been sufficient to facilitate insulin secretion. Second, some
arachidonate release must occur soon after stimu- lation which is independent of cyclooxygenase products or substrate would never become availa- ble to the cyclooxygenase. It is possible that effects of 12-lipoxygenase products on insulin secretion are confined to this early, indomethacin-insensitive interval. Third, indomethacin (unlike ETYA) com-
pletely inhibited the synthesis of cyclooxygenase
products, and it is possible that smaller amounts
of 12-lipoxygenase products are required to facili-
tate secretion in the absence of cyclooxygenase products than in their presence.
We are aware of little previous work examining the effect of ETYA on glucose-induced insulin
secretion from isolated islets, but the structurally
dissimilar lipoxygenase inhibitors nordihydroguai-
aretic acid and BW755C have been reporeted to
suppress glucose-induced insulin secretion from
isolated islets [37,47]. The lipoxygenase inhibitor
BW755C has also been reported to suppress arachidonic acid-induced insulin secretion from
cultured pancreatic cells from rat neonates, and exogenous 12-HPETE has some insulin secreta-
gogue activity on such preparations [21]. None of these studies examined islet synthesis of lipo-
xygenase products from endogenous arachidonate or how this synthesis was affected by glucose or a
lipoxygenase inhibitor. The cyclooxygenase inhibi-
tors indomethacin and flurbiprofen have been re- ported by other workers not to influence glucose-
induced insulin secretion from isolated islets
[8,37,47]. It is of interest that 12-HPETE synthesis
stimulates glucose oxidation in platelets indirectly
due to oxidation of NADPH and activation of the hexose monophosphate shunt which occur conse-
quent to reduction of 12-HPETE to 12-HETE
[48,49]. Prevention of 12-HPETE synthesis by
ETYA reduces glucose oxidation in platelets [48,49]. If similar effects occur in islets, they may
relate to the inhibition of glucose-induced insulin
secretion by ETYA.
Further evaluation of the possibility that arachidonate metabolites participate in glucose-in-
duced insulin secretion by isolated islets will re- quire examination of the effects of additional in-
hibitors of arachidonate metabolism on insulin secretion and on metabolite synthesis, examination of the time course and dose-response relationships of these effects, and examination of the time course and dose-response relationships of insulin secre- tion provoked by exogenous standards of the arachidonate metabolites which have been identi- fied as islet products, including 12-HPETE and 12-HETE. These studies should be feasible with the analytic approaches described above. One pos-
sibility that requires evaluation is that the effects
of arachidonate lipoxygenase inhibitors on insulin
secretion are not mediated by inhibition of
lipoxygenases but rather by inhibition of other
processes, such as cytochrome P-450-dependent oxygenation of arachidonate. Products of the latter
pathway have been reported to provoke insulin
secretion from isolated islets, and lipoxygenase inhibitors have been reported to suppress forma-
tion of these compounds in other tissues [26]. Whether islets are capable of synthesizing these
compounds remains an open question, although
initial attempts at demonstrating their synthesis by
islets have been unsuccessful [26].
Acknowledgements
This work was supported in part by a grant to M.L.M. from the National Institutes of Health
(AM 06181) and by grants to J.T. from the Washington University Diabetes Research and
Training Center (NIADDK P60 AM20579), the
Washington University Biomedical Research Sup-
port Grants Program (NIH BSRG SO7 RR 05389)
the Pharmaceutical Manufacturer’s Association
Foundation, the Juvenile Diabetes Foundation and the National Institutes of Health (AM 34388). The
excellent technical assistance of Mark Pautler,
Richard Thoma, Deirdre Buscetto and C. Joan Fink has been greatly appreciated, as has the
interest and advice of Dr. Jay McDonald, Dr. Paul
Lacy and Dr. Philip Needleman. Special thanks are due to Paula Game1 and to Jane Huth for the
typing of the manuscript. We are grateful to Dr.
Aubrey Morrison for providing access to the Hewlett-Packard 5985B mass spectrometer within
the Washington University Mass Spectrometry
Resource Center (NIH RR 00954).
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