Arachidonic acid metabolism in isolated pancreatic islets. III. Effects of exogenous lipoxygenase...

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BBA 51853

Arachidonic acid metaboku in isolated pancreatic islets. III. Effects of

exogenous lipoxygenase products and inhibitors on insulin secretion

J&n Turk a-b, * , Jerry R. C&a b,*4 and Michael L. McDaniel ’

(Received August 8th, 1984) (Revised manuscript October 25th, 1984)

Key words: Arachidonate metabolism; ~ydroxyei~os~no~~ acid; Lipoxygenase; Insulin secretion; (Rat pancreatic islet)

Isolated pancreatic islets from the rat have been demonstrated by stable isotope dilution-mass spectrometric methods to synthesize the ltlipoxygenase product 12-hydroxyeicosatetraenoic acid (1%HE’I’E) in amounts of 13 to 2.8 ng per lo3 isIets, No de&e&able amoamts of 5-HETE and only trace amounis of 15-HETE could be ~~~~~~ by these methods. No~hyd~~ai~etic acid (NDGA) and BW755C have been demonstrated to inhibit isfet t2-HETE synthesis and aIso to inhibit gtncose-induced insulin secretion. Inhibition of insulin secretion and of 12”HETE synthesis exhibited simiiar dependence on the concentration of these compounds. Eicosa-5,8,11,14-tetrynoic acid (ETYA) also inhibited glucose-induced insulin secretion, as previously reported+ at concentrations which inhibit islet 12-HETE synthesis. Exogenous 12-HEm partially reversed the suppression of ~uc~-i~~ insulin see&ion by fipoxygenase i~ibito~ but exogenous 12-hy~~roxye~~~e~~oic acid (IZHPETE), 15-HPETE, %iPETE, l§HETI$ or 5-HETE dii not reverse this suppression. These observations argue against the recently suggested hypothesis that islet synthesis of 5-HETE modulates insulin secretion. Suppression of glucose-induced insulin secretion by ETYA, BW755C and NDGA may be due to inhibition of the islet 12Gpoxygenase by these compounds. The possibility that other processes invofved in glucose-induced insulin secretion are inhlMted by E’IYA, BW755C and NDGA tannot yet be excluded.

Introduction

Recent observations from this group [1,2] indicate that intact pancreatic islets isolated from adult

* To whom correspondence and reprint requests should be addressed at Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.

*+ Present address: The Upjohn Company, Kalamazoo, MI, U.S.A.

Abbreviations: HETE, hydro~y~j~o~tetraenoic acid; ETYA, eicosa-5,8,11,14-tetrynoic acid; HPETE, hydroperoxyeicosatetraenoic acid; RP, reversed phase; NP, normal phase; El, electron impact; CI, chemical ionization; NI, negative ion; PI, positive ion; ME, methyl ester; PFBE, pentafluorobenzyl ester; TMS, tr~methyisiIy1; NDGA, nordihy~ro~u~~etic acid.

rats convert endogenous arachidonic acid to four major cyclooxygenase products and to the lipoxygenase product 1ZHETE. Quantitatively, 12- HETE is the most abundant of these products j1,2& Isolated islets incubated with 28 mM glucose secrete more insulin and produce larger quantities of 12-HETE and of the cyclooxygenase products than do islets incubated with 3 mM glucose i2]. The glucose-induced production of f Zlipo- xygenase products may participate in glucose-induced insulin secretion, since inhibition of the islet 1Zlipoxygenase and cyclooxygenase enzymes with eicosa-5,8,11,14-tetryrmic acid (ETYA) suppresses glucose-induced insulin secretion. Inhibition of the islet cyclooxygenase with ~ndomethacin does not

~~-2~6~~8~/~~3.~~ 0 1985 Etsevier Science Publishers B.V. (Biomedical Division)

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influence insulin secretion [2]. The concentration

of ETYA (20 PM) required to inhibit glucose-in-

duced insulin secretion by 63-74s results in 88%

suppression of islet 12-HETE synthesis [2], indicat-

ing a reasonably close correspondence between the

two phenomena for this agent. Others have dem-

onstrated that ETYA also inhibits insulin secre-

tion by cultured pancreatic cells from rat neonates

and that 12-HETE is synthesized by these cultured cells [6].

Additional compounds which may inhibit

arachidonate lipoxygenases, including nordi-

hydroguaiaretic acid (NDGA) and BW755C, also inhibit insulin secretion from cultured pancreatic

cells from rat neonates [3-71, isolated pancreatic

islets [S-lo] or perfused pancreas preparations [ll]. NDGA and BW755C have been shown to

impair conversion of ’ H-labelled arachidonate to ‘H-labelled 12-HETE at concentrations which in-

hibit insulin secretion by cultured pancreatic cells

from rat neonates [4,6]. It is not certain that

12-HETE synthesized by these cultured neonatal

cells arises from endocrine cells rather than from

the predominant cell type in the cultures (fibrob- lasts). In addition, discrepancies have been re-

ported for the effects of inhibitors on the synthesis

of products from exogenous, radiolabelled arachidonate versus endogenous arachidonate [2].

It has not yet been demonstrated that NDGA and BW755C inhibit the conversion of endogenous

arachidonate to lipoxygenase products by intact isolated islets at concentrations which suppress

glucose-induced insulin secretion. In part this re-

flects the technical difficulty in quantitating islet lipoxygenase products in the amounts synthesized

from endogenous arachidonate. We have examined the concentration dependence of the in-

hibition of the islet 12-lipoxygenase by BW755C

and NDGA with a recently developed mass spec-

trometric measurement technique [I] and have compared this to the concentration dependence of the inhibition of insulin section by the two agents.

The effect of lipoxygenase inhibitors to suppress insulin secretion has been attributed to the participation of various oxygenated metabolites of arachidonate acid in stimulus-secretion coupling, including products of the 12-lipoxygenase [6,7], of the 5-lipoxygenase [9,12], or of non-lipoxygenase pathways sensitive to these inhibitors [lo]. Involve-

ment of specific compounds within those groups

of metabolites has been inferred from the secreta-

gogue effects of such compounds obtained from

exogenous sources and incubated with pancreatic

cell cultures [6,7] or isolated islets [9-111. The

synthesis of many of these compounds, including 5-lipoxygenase products, from endogenoux arachidonate by isolated islets has never been demonstrated, however. We have attempted to de-

tect and quantitate islet synthesis of 5-HETE and of 15-HETE relative to 12-HETE by mass spectro-

metric methods in order to evaluate the possible

participation of these compounds in insulin secre-

tion.

The potential insulin secretagogue effects of the

primary lipoxygenase products 12-HPETE and 5- HPETE have not previously been examined with

isolated islets. To further evaluate the possible

participation of 12- and/or 5-lipoxygenase products in insulin secretion, we have prepared syn-

thetic, highly purified 12-HPETE and 5-HPETE and have examined their ability to elicit or potenti-

ate insulin secretion and to reverse the suppression

of insulin secretion by lipoxygenase inhibitors.

Materials and Methods

Muterials

The following materials were obtained from

New England Nuclear (Boston, MA): [5,6.8,9,11,

12,14,15-‘H,]Arachidonic acid (100 Ci/mmol); 12-[5,6,8,9,11,12,14,15-3H,]Hydroxy-5,8,10,l4-eico-

satetraenoic acid (‘H,-12-HETE, 60 Ci/mmol);

5-[5.6,8,9,11,12,14,15-3H,]hydroxy-6,8,11,14-eicosatetraenoic acid ( 3H,-5-HETE, 60 Ci/mmol).

Unlabelled arachidonic acid was obtained from NuCheck Prep (Elysian, MN). All organic solvents

were obtained from Burdick and Jackson Labora- tories (Muskegon, MI). Eicosa-5,8,11,14-tetrynoic acid (ETYA) was obtained from Hoffman LaRoche (Nutley, NJ) through the courtesy of Dr. James Hamilton. Nordihydroguaiaretic acid and esculetin were obtained from Sigma Chemical (St. Louis, MO). BW755C was obtained from the Wel- lcome Research Laboratories (Kent, England) through the courtesy of Dr. P.J. McHale. Male Sprague-Dawley rats (180-200 g body weight) were obtained from Sasco (O’Fallon, MO). Collagenase Type IV was obtained from Worthington. Tissue

25

culture medium (CMRL 1066) was obtained from Gibco (Grand Island, NY). Pentax bovine albumin (fatty acid-free, fraction V) was obtained from Miles Laboratories. N, ~-Dimethylacetamide and tetramethylammonium hydroxide were obtained from Matehson, Coleman and Bell (Norwood, Ohio). Pentafluorobenzylbromide and N,O-bis(trimethylsilyl)trifluoroacetamide were obtained from Pierce (Rockford, IL).

Ex~racriun. Arachidonate metabolites were re- covered from aqueous solutions by addition of 2 vol methanol, acidification to pH 3.5 with 0.5 M HCl, and extraction with 1 vol. CH,CI, twice. The combined CH,CI, extracts were washed with one fifth volume of water, dried over Na,SQ,, and then (a) subjected to HPLC analysis, (b) derivatized, or (c) stored in CH,Cl, at -70°C.

Liquid chromatography. High-performance liquid chromatography (HPLC) was performed on a Varian Model 8500 instrument (Varian, Walnut Creek, CA) in the reversed phase (RP) or normal phase (NP). The following columns were obtained from Waters Associates (Milford, MA): I (PBondapak Cts, 3.9 mm X 30 cm), II (PPorasil, 3.9 mm x 30 cm), III (~Bondapak C,,, 7.8 mm x 30 cm), and IV (FPorasil, 7.8 mm x 30 cm). Col- umn V consisted of two BioSil ODS-SS (250 mm X 4 mm) columns in series obtained from BioRad Laboratories (Richmond, CA). Thin-layer chromatography (TLC) was performed on Analtech (Newark, DE) Uniplates (Silica GF, 2.5 X 10 cm, 250 micron). The following solvent mixtures (v/v) were employed: A (methanol/water/acetic acid, 75 : 25 : 0.01); B (a linear gradient from hexane/ acetic acid, 100 : 0.8, to CHCl,/acetic acid, 100: 0.8, over 120 min); C (acetonitrile/water/ acetic acid, 50 : 50 : 0.1); D (hexane/isopropanol/ acetic acid 100 : 0.8 : 0.1); E (hexane/

isopropanol/acetic acid, 100 : 0.8 : 0.1); F (diethyl ether/hexane/acetic acid, 55 : 45 : 1); G (chloro- form/acetic acid/water, 5 : 5 : 1); H (hexane/ isopropanol/ acetic acid, 100 : 1.75 : 0.1); I (acetonitrile/water/acetic acid, 45 : 55 : 0.1); and J (acetonitrile/water, 70 : 30).

Deriuatization. The delta lactone of 5-HETE was hydrolyzed in (v/v) triethylamine/pyridine/ water 1 : 10: 10, for 30 min at room temperature

[22]. Carboxyl groups were converted either to (a) methyl esters (ME) with ethereal diazomethane or to (b) pentafluorobenzyl esters (PFBE) by any of a variety of methods [23-281, the most satisfactory of which [28] employed treatment with 20 ~1 of a solution (v/v) of N, N-dimethylacetamide/ tetramethyl ammonium hydroxide/ methanol (8 : 5 : 15) and 20 ~1 of a solution of (v/v) pentafluorobenzyl bromide/dimethyl acetamide (1: 3) for 30 min at room temperature, followed by concentration to dryness under nitrogen, recon- stitution in water and extraction with CHzCl,. Hydroxyl groups were converted to the trimethylsilyl ethers with N,O-bis(trimethylsilyl)trifluoroacetamide in pyridine for 30 min at room temperature. Hydroperoxides were reduced to hydroxyl groups with triphenylphosphine (Fisher, St. Louis, MO) in diethyl ether as described [17] or with NaBH, (Fisher) in methanol as described [15].

Gas chromatography-mass spectrometry. Gas chromato~aphy was performed on a Hewlett Packard 5840A gas ~hromatograph interfaced with a Hewlett Packard 5985B mass spectrometer. A standard packed column (i.d. 2 mm, 2 ft length, 2% OVlOl on Supelcoport) (Supelco, Bellefonte, PA) was operated isothermally at 210°C or at 225°C. A capillary column (Hewlett Packard Ul- traperformance Capillary column, 25 m length, crosslinked methylsilicone, i.d. 0.31 mm, film thickness 0.17 pm) was operated with a Grob-type injector in the splitless mode with helium as carrier gas (inlet pressure 20 lb/in2, injector temperature 250°C). The capillary column was programmed from 85°C to 225°C or to 240°C at a rate of 30°C per min. Quantitation of islet-derived HETE compounds relative to deuterated internal standards was performed on the capillary column. Under the indicated conditions, the derivatives PFBE, TMS of the HETE compounds exhibited a retention time of about 14.9 min, and the regional isomers were not clearly separated from each other. Quantitative analyses were performed with the mass spectrometer in the negative ion-chemical ionization (MI-CI) mode with methane as reagent gas (source pressure 2 - 10e4 tori-, ionization volt- age 230 eV). This mode of analysis confers great sensitivity [23-271. The negative-ion methane chemical ionization mass spectra of the TMS derivatives of the pentafluorobenzyl esters of the

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HETE compounds exhibited a prominent ion at

(m/z) M - 181, corresponding to the loss of the

pentafluorobenzyl group. Selected ion monitoring

of the ion pair 391 vs. 399 was therefore used to

quantitate endogenous islet-derived HETE com-

pounds relative to the deuterated internal stan-

dards. Structural analyses of the standard com-

pounds were performed with the mass spectrometer in the positive-ion (PI))electron impact (EI)

mode in order to obtain an informative fragmenta-

tion pattern.

Preparation of standards. 15(S)-[5,6,8,9,11,12, 14,15-3H,]Hydroxy-5,6,11,13-eicosatetraenoic acid

(15(S)-[‘H,]HETE) was prepared by NaBH, reduction of [‘H,]hydroperoxy-5,8,10,13-eicosate-

traenoic acid (15(S)-[‘H,]HPETE) as described

[13,14]. The 15(S)-[‘H,]HPETE was prepared by

the action of soybean lipoxygenase (type IV, Sigma

Chemical, St. Louis, MO) (linoleate : oxygen

oxidoreductase, EC 1.13.11.12) on [7Hx]arachi-

donic acid [13]. The IS(S)-[‘H,]HETE was puri-

fied by RP-HPLC (column I, solvent A, retention

vol. 14-16 ml). A similar procedure was used to

prepare unlabelled 15(S)-HETE. This material was

quantitated after purification by its ultraviolet ab-

sorbance at 235 nm [15] in methanol as de-

termined on a GCA McPherson (acton, MA)

Model EU-700 spectrophotometer. [5,6,8,9,11,12,14,15-2H~]Arachidonic acid

([ * H,]arachidonic acid) was prepared from ETYA and deuterium gas (MG Scientific Gases, Chicago

IL) [16]. The compounds 15-[‘H,]HETE, 12-

[’ HJHETE and 5-[ ‘H,]HETE were prepared

from [ 2Hx]arachidonic acid with H,O, (Fisher

Chemical, St. Louis, MO) and CuCIZ (Fisher) and purified by SP-HPLC (column IV, solvent B) and

then RP-HPLC (column III, solvent A) essentially

as described [IS]. The purified compounds were quantitated [15] and assigned a diene geometry

[17] by ultraviolet absorption spectroscopy. The regional isomers of the [‘H,]HETE compounds were distinguished by GC (OVlOl, temperature 225C, Cv21.3)-MS (PI-EI) and analyses of the ME, TMS derivatives. All isomers exhibited ions (m/z) at 414 (M), 399 (ML 15, loss of CH,), and 383 (M - 31, loss of OCH,). In addition only the mass spectrum of 12-[‘H,]HETE (ME, TMS) exhibited a prominent ion at m/z 301; only that of 15-t *H,]HETE (ME, TMS) at m/z 229 and 343;

and only that of 5-[‘H,]HETE at m/z 204. 258. and 313.

15-HPETE, 12-HPETE and 5-HPETE were

prepared from arachidonic acid by air auto-oxida-

tion [17] for 48 h. The mixture of monohydroper-

oxides was separated from unreacted arachidonic

acid and multiply oxygenated species by RP-HPLC

(column III, solvent C) of 50 mg aliquots of the

crude, concentrated reaction mixture. Fractions with elution volumes between 70 and 140 ml were

pooled and extracted with CHzCl,. The con-

centrated extract was subjected to NP-HPLC (col-

umn IV, solvent D for 128 ml, followed by a linear gradient over 25 min to solvent E. then solvent F

for 100 ml). The 12-HPETE and 15-HPETE eluted

between 72 and 88 ml, were poorly resolved from each other and were collected together. The 5-

HPETE eluted between 220 and 232 ml and was collected separately. The 12-HPETE and 15-

HPETE were separated from each other by RP- HPLC (column III, solvent C). The elution volume

for 15-HPETE was 988110 ml and that of 12-

HPETE was 112-120 ml. TLC analysis (solvent F)

of the products revealed single spots for 15-HPETE

(R,: 0.60). 12-HPETE (R, 0.60) and 5-HPETE

(R,. 0.50) that could be visualized either with iodine vapor or with a peroxide spray containing

0.1% N, N-dimethyl-p-phenylenediamine (Sigma) in

solvent G [18]. Treatment of these materials with triphenylphosphine or NaBH, slightly reduced TLC R, values and resulted in loss of color devel- opment with the peroxide spray reagent. GC

(OVlOl. 225”C)-MS(PI-EI) analysis of the derivatives (ME, TMS) of the HETE compounds pre-

pared from reduction of the HPETE isomers revealed a carbon value of 21.3, relative to a series of

saturated fatty acid methyl ester and ions at (m/z) 406(M),391(M-15,lossofCH,),375(M-31.

loss of OCH,) and 316 (M - 90, loss of ((CH,),SiOH). The mass spectrum only of 12- HETE (ME, TMS) exhibited a prominent ion at 295 (M- 111, loss of (CH)z(CHz),CH,). The mass spectrum only of 15-HETE (ME, TMS) exhibited prominent ions at 225 ( A4 - 181. loss of

(CH),CH,(CH),(CH,),- CO,CH,) and 335 (M - 71, loss of (CH,),CH,). The mass spectrum of only 5-HETE (ME, TMS) exhibited prominent ions at 302 (M - 203, loss of

(CH,),SiOCH(CH,),CO,CH,) and 255 (M -

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151, loss of CH,(CH),CH,(CH),(CH,),CH,). NP-HPLC (column IV, solvent D) analysis dis-

tinguished the purified 12-HPETE (retention vol. 72 ml) from its triphenylphosphine reduction

product 12-HETE (retention vol. 56 ml) and dis-

tinguished 15-HPETE (retention vol. 76 ml) from

its triphenylphosphine reduction product 15-HETE

(retention vol. 60 ml). 15-HETE prepared in this

way co-eluted with 3H-labelled 15-HETE prepared

with the soybean lipoxygenase, and 12-HETE prepared in this was co-eluted with 14C-labelled 12-

HETE prepared with human blood platelets [l].

NP-HPLC analysis (column II, solvent H) also distinguished 5-HPETE (retention vol. 16 ml) from

its triphenylphosphine reduction products 5-HETE

(retention vol. 26 ml). 5-HETE prepared in this

way co-eluted with 5-[14C]HETE prepared with

human blood leukocytes [l] and with SHETE

prepared via 6-iodo-5-hydroxy-8,11,14-eico-

satrienoic acid S-lactone by the method of Hub-

bard et al. [20]. The purified HPETE isomers were

quantitated by ultraviolet absorption spectrophotometry [19] and stored in CH,Cl, at -70°C.

Each HPETE compound was analyzed by TLC

and NP-HPLC just before use and was re-purified

by NP-HPLC if more than 5% decomposition to

the HETE, to the truns-truns isomer [21], or to

other compounds had occurred.

Isolation and culture of islets. Pancreatic islets

were obtained under aseptic conditions from rats fed ad libitum. The islet isolation procedure is

described in detail elsewhere [29-311 and involved disruption of pancreatic acinar tissue by instilla-

tion of buffer via the cannulated bile duct, dissec-

tion and excision of the distended pancreas, re- moval of fat and adherent lymph nodes, mincing,

collagenase digestion and centrifugation over dis-

continuous density gradients of Ficoll in water.

The isolated islets were then washed in tissue

culture medium and selected under a stereo-micro-

scope to exclude any contaminating tissues. The islets were then used immediately or cultured over-

night under an atmosphere of 95X sir/5% CO, at 24°C in tissue culture medium CMRL 1066 containing 8 mM D-glucose, 1% L-glutamine, 10% heat-inactivated fetal bovine serum, 0.5% penicillin and 0.5% streptomycin.

Isolation of rat platelets. Platelets were isolated from anticoagulated (EDTA) rat blood as de-

scribed in detail elsewhere [l]. In brief, the proce-

dure involved low speed (100 X g, 10 min) centrifugation to remove red cells and higher speed

centrifugation (2000 x g, 6 min) to (a) pellet the

platelets from plasma and (b) then to wash the

platelets with calcium-free Krebs-Ringer bi-

carbonate buffer (115 M NaCl/S.O mM KC1/24

mM NaHCO,/l mM MgCl,) saturated with 95%

sir/5% CO, and supplemented with 3 mM glu-

cose. Platelets were counted and assessed for con-

tamination as described elsewhere [ll]. Platelet

production of arachidonate metabolites in the

presence and absence of inhibitors was assessed

after incubation under conditions indentical to

those described below for islets. Incubations were

initiated by addition of calcium chloride (final

concentration 2.5 mM), additional glucose (final

concentration 28 mM), and calcium ionophore

A23187 (final concentration 10 PM). Metabolites

were extracted as described above and analyzed by

RP-HPLC (column I, solvent I) with flow-through

ultraviolet monitoring at 235 nm.

Incubation of islets. For studies examining the

influence of various compounds on arachidonate

metabolism, isolated islets (approx. 1.5 X 104) were

pre-incubated (30 min, 37°C) in medium (5 mM

Hepes/135 mM NaC1/24 mM NaHCO,/5 mM

KCl/ 1 mM MgCl,/2.5 mM CaCl, (pH 7.4))

supplemented with glucose (3 mM) with or without

the test compound. The islets were then collected

by centrifugation and resuspended in fresh medium

(2 ml of the same composition (twice) and then divided into two to four groups in individual

siliconized tubes. Incubations were initiated by the

addition of glucose (final concentration 28 mM) and were continued for 30 min at 37°C. At the

end of this period, a mixture of 3 H- and ’ H-labelled

internal standards of 5-HETE, 15-HETE and/or

12-HETE was added in methanol (1 vol.). Par-

ticulate matter was removed by centrifugation (3000 X g, 5 min). Insulin in the pellet was ex-

tracted with 75% ethanol/l.5% HCl and measured

by radioimmunoassay [33], as was the insulin con- tent of an aliquot of the supernatant to insure that the various samples contained islet masses differ- ing by less than 7% as described previously [2]. The remainder of the supernatant was acidified to pH 3.5 (1 M HCl) and extracted twice with CH,Cl, (1 vol.). The HETE compounds in the extract were

2x

concentrated to dryness (N,). converted to the

pentafluorobenzyl esters, analyzed by RP-HPLC

(column V, solvent J). extracted from the RP-

HPLC solvent, converted to the trimethylsiIy1 es-

ters, and quantitated by CC-MS as described

above.

For studies examining the effects of inhibitors

of arachidonate metabolism on insulin secretion,

20 islets were randomly selected under a stereomi-

croscope for each sample. The islets were then

pre-incubated (30 min, 37°C) in albumin-free

medium (of the composition described above) con-

taining 3 mM glucose and the desired concentra-

tion of inhibitor. At the end of the preincubation

period, the medium was removed and replaced with fresh medium containing glucose (3 or 28

mM) and the same concentration of inhibitor used in the preincubation period. The experimental in-

cubation then proceeded for 30 min at 37°C and

was terminated by addition of medium (200 ~1)

containing bovine serum albumin (0.2%), mixing

and withdrawal of an aliquot for the radioim-

munoassay of secreted insulin.

For studies examining the effects of exogenous

HETE or HPETE standard compounds on insulin

secretion, a protocol similar to that described above

was employed. The test compound was added after the pre-incubation period and washing, and

the addition constituted the beginning of the ex-

perimental incubation period. Solutions of test compounds. Aqueous solutions

of the HPETE and HETE compounds were prepared just before use. The desired amount of the

test compound was transferred to a 1 ml Reacti- vial (Pierce) in ethanol, blown to dryness under N, and reconstituted in 15 ~1 of ethanol. 9 vol. (135

~1) of incubation medium were then added. 10 (-11

of the resultant solution were added to each batch

of islets exposed to the test compound to initiate the incubation. 10 ~1 of a solution of ethanol (15 ~1) and incubation medium (135 ~1) were added to control incubations not exposed to the test compound. Complete dissolution of the HPETE and HETE compounds under these conditions was demonstrated with ultraviolet spectrophotometry. HPLC analyses indicated that the HPETE and HETE compounds were stable in solution in the incubation medium for up to 3 h under these conditions. Arachidonic acid was dissolved in 0.1

M Na &O, (1 mg/ml).

BW755C was dissolved in dimethylsulfoxide/

0.15 M NaCI, 1 : 17 (v/v) at a concentration of 5.1

mg/ml. NDGA was dissolved in dimethylsulfo-

xide/O.lS M NaCl, 1 : 3 (v/v) at a concentration

of 1.75 mg/ml. ETYA was dissolved in 0.1 M

Na,CO, at a concentration of 0.6 mg/ml. Escule-

tin was dissolved in dimethylsulfoxide/0.15 M NaCl, 1 : 1 (v/v) at a concentration of 10 mg/ml.

Indomethacin was dissolved in 0.1 M Tris buffer

(PI-I 8.2)/l N NaOH, 500 : 1 (v/v) at a concentra-

tion of 10 mg/ml. All of these compounds were

then diluted into incubation medium to achieve

the desired concentration. The appropriate vehicle was added to control incubations which did not

receive an inhibitor.

Results

It has recently been proposed that 5-HETE modulates insulin secretion from isolated pan-

creatic islets based on the reported insulin

secretagogue effects of exogenous 5-HETE at concentrations of 25 PM-100 PM [9]. We have ob-

served formation of 12-[3H,]HETE but not 5-

[“H,]HETE or 15-[3H,]HETE by isolated islets

incubated with [3H,]arachidonic acid [1,2], which

raises doubt about the ability of islets to synthesize

5-HETE. We have observed several discrepancies

between the metabolism of exogenous, radio-

labelled arachidonate and endogenous arachidonate by isolated islets [1,2], however. and we

therefore performed quantitative mass spectrometric measurements of islet-derived 12-HETE, 5-

HETE and 15-HETE synthesized from endogenous arachidonate (Fig. 1). Isolated islets were incubated with a maximally stimulatory con-

centration (28 mM) of glucose. A mixture of ‘H-

and ‘H-labelled internal standards of 12-. 5- and

1%HETE was then added, extracted along with

any of the endogenous (protium) form of the molecules present in the incubation mixture, converted to the pentafluorobenzyl esters, separated by HPLC, derivatized and analyzed by GC-MS. Synthesis of 1ZHETE was clearly demonstrabie under these conditions (Fig. 1, panel C) and amounted to about 1.7-2.8 ng per lo3 islets (mean 2.3 ng). (Assuming a mean intracellular water volume of about 3 nl per islet [34], diluting the measured mass of 12-HETE into the intracellular

29

A HPLC ANALYSIS OF HETE ISOMERS

Cl8 RP-HPLC

ACCN 7WH20 30

D. GC-MS ANALYSIS OF ISLETS

S-HETE (PFBE, TMSJ

117r

Area: 261 I

ELUTlON TIME (Mid

GC-MS ANALYSIS OF ISLETS

~HETE (PFBE, TMS)

Area: 24854

14 15 16 17 ld 15 16 17

ELUTION TIME (Mu-t) ELUTION TIME (Mtn)

Fig. 1. Mass spectrometric analysis of hydroxyeicosatetraenoic acid (HETE) isomers synthesized from endogenws arachidonic acid by isolated pancreatic islets. For each experiment pancreatic islets (approx. 1.5. 104) were isolated from 30 rats as described in the Methods section, suspended in KRB medium containing glucose (3 mM), and divided into one to four equal populations. Each population was then incubated for 30 min at 37% with shaking in KRB medium (see Methods section) containing 3 mM glucose with or without an inhibitor of arachidonic acid metabolism. At the end of this pm-incubation period, the islets were collected by centrifugation and resuspended in fresh KRB medium containing 28 mM glucose with or without an inhibitor of arachidonic acid metabolism. The islets were then incubated for 30 min at 37°C with shaking. At the end of this experimental incubation period, a mixture of 3H- and 2H-labeiled internal standards of ES-HETE, 12-HETE and 5-HETE was added in methanol (1 vol.). The products were extracted, concentrated and converted to the pentafluorobenzyl ester (PFBE) derivatives as described in the Methods section. The derivatized produets were then analyzed by RF-HPLC (column V, solvent J). Panel A illustrates the separation of the PFBE derivatives of 15-HETE, 12-HETE and 5-HETE under these conditions. The radioactive peaks derive from the ‘H-labelled component of the internal standards. Each of these peaks was collected separately. The PFBE-HETE compounds were extracted from the RP-HPLC solvent with CH,C12, concentrated and converted to the trimethylsilyl ether (TMS) derivatives as described in the Methods section. Each derivatized HETE isomer was then analyzed by capillary column gas chromato~aphy-negative-ion methane chemical ionization-mass spectrometry with selected monitoring of ions at m/z 399 and m/z 391 as described in the Methods section. The ion at m/z 399 originates from the 2H-labelled internal standard HETE derivative, and the ions at m/z 391 originates from the derivatized endogenous HETE isomer. Panel B is the ion-current vs. elution time tracing for the derivatized 15-HETE, panel C that for 12-HETE, and panel D that for 5-HETE. Similar tracings were obtained in each of five separate experiments.

water would yield a concentration of about 2.5 PM 12(S)-HETE. Only trace amounts of islet-derived 15HETE could be detected (Fig. 1, panel B), and no islet-derived 5-HETE could be detected (Fig. 1,

panel D). The mass spectrometric measurements therefore supported the results of the studies [1,2] with radiolabelled arachidonate which indicated that 124poxygenase products, but not 5- or 15

lipoxygenase products were formed in substantial amounts by isolated pancreatic islets.

As illustrated in Table I, the Iipoxygenase and cyclooxygenase inhibitor eicosatetrynoic acid (ETYA) suppressed glucose-induced insulin secretion from isolated islets. Coupled with the observation above that the 1Zlipoxygenase appears to be the principal Iipoxygenase active in isolated

30

TABLE I

INFLUENCE OF EXOGENOUS 12-HYDROPEROXY-

EICOSATETRAENOIC ACID (12-HPETE) ON INSULIN

SECRETION FROM ISOLATED PANCREATIC ISLETS

Pancreatic islets were isolated from ten rats on each of four

separate days, and the islets were suspended 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

pre-incubated for 30 min at 37’C with shaking in incubation

medium (200 pl) containing 3 mM glucose with or without an

inhibitor. The preincubation medium was then removed and

replaced by medium (200 ~1) containing glucose (3 or 28 mM)

with or without an inhibitor. Incubations were initiated hy

addition of an aliquot (10 ~1) of diluent (see Methods section)

containing no other additives (controls) or containing the de-

sired concentration of the fatty acid derivatives to be tested as

potential secretagogues. Incubations were then continued for

30 min at 37°C. At the end of this interval, an aliquot (200 ~1)

of bovine serum albumin (0.2%) in incubation medium was

added and aliquots of the experimental medium were withdrawn

for the radioimmunoassay of insulin. Each sample was mea-

sured in triplicate and three separate incubations were per-

formed at each experimental condition in each experiment.

Tabulated secretion rates represent means, and standard errors

of the means are indicated (n = 4). When inhibitor was present

it was 20 PM; when potential secretagogue was present it was 5

PM.

Glucose Inhibitor Potential Insulin

concentration secretagogue secretion

(mM) (pU/min per islet)

3 None None 1.58 * 0.33

8 None None 3.23 + 0.77

28 None None 6.12kO.46

3 ETYA None 1.48 + 0.29

8 ETYA None 3.36 + 0.98 28 ETYA None 2.67 + 0.46

3 None I2-HPETE 1.38 * 0.34

8 None 12-HPETE 3.29 i 0.33

28 None 12-HPETE 5.67 + 0.64

3 ETYA I2-HPETE 1.22 + 0.28

8 ETYA 12-HPETE 3.02 i 0.54

28 ETYA 12-HPETE 2.33 kO.99

islets and with the observation that the selective cyclooxygenase inhibitor indomethacin does not influence insulin secretion [2], the suppression of secretion by ETYA suggests that a 124ipo- xygenase product might participate in glucose-in-

duced insulin secretion. The potential insulin

secretagogue effects of the primary 12-lipo-

xygenase products 12-HPETE were therefore ex-

amined. At a concentration of 5 PM (2.5 PM each

of S and R enantiomers) racemic 12-HPETE did

not influence insulin secretion at glucose con-

centrations of 3 mM (basal secretion), 8 mM (about

half-maximal stimulation), or 28 mM (maximal stimulation) (Table I). Suppression of glucose-in-

duced secretion by ETYA also was not reversed by

5 PM 12-HPETE (Table I). Lower concentrations (50 nM-1.5 PM) of 12-HPETE also failed to

influence insulin secretion, although 50 PM 12- HPETE suppressed glucose-induced insulin secre-

tion by about 50% (data not shown). The suppres- sive effect of 50 PM 12-HPETE appeared to be

TABLE II

INFLUENCE OF EXOGENOUS

TETRAENOIC ACID (12-HETE)

12-HYDROXY EICOSA- ON INSULIN SECRE-

TION FROM ISOLATED PANCREATIC ISLETS

Conditions were as described in Table I except that the fatty

acid derivative tests as a potential secretagogue was 12.hy-

droxy-eicosatetraenoic acid (12-HETE) rather than 12-HPETE. When ETYA was present it was 20 PM.




3 None

28 None

3 ETYA

28 ETYA

3 None

28 None

3 ETYA

28 ETYA

3 None

28 None

3 ETYA

28 ETYA

None

1.16rf-0.21

6.44 + 0.34

None 0.97iO.18

None 2.85 *0.45

12-HETE

(5 LLM) 12-HETE

(5 pM)

0.92+0.13

6.53 f 0.70

12-HETE

(5 PM) 12-HETE

(5 PM)

1.18kO.24

3.81 + 0.64

12-HETE

(50 PM) 12-HETE

(50 pM)

1.04+0.14

4.78 + 1.06

12-HETE

(50 PM) 12-HETE

(50 PM)

1.07 + 0.21

2.84+ 1.13

non-specific, since similar effects were seen with

all tested fatty acid derivatives at this concentra-

tion (see below). The metabolic reduction product of 12-HPETE

is 12-HETE, and the potential insulin secretagogue

effects of 1ZHETE were also examined. At a

concentration of 5 PM, racemic 1ZHETE did not

influence insulin secretion at glucose concentrations of 3 or 28 mM (Table II). The suppression of

glucose-induced insulin secretion was partially reversed by 5 PM IZHETE (Table II). This effect

was statistically significant by paired, two-tailed t

analysis: in the presence of 20 PM ETYA, the

secretion rate at 28 mM glucose in the presence of 5 PM 12-HETE was greater than that in the

absence of 12-HETE by 0.96 + 0.29 &J/min per

,-n 082 ” 0” u 0”

ELUTION VOLUME lmll

Fig. 2. Concentration dependence of inhibition of the rat

platelet cyclooxygenase and 12Jipoxygenase by BW755C.

Platelets were isolated from anticoagulated rat blood as de-

scribed in the Methods section and incubated with the desired

concentration of BW755C (O-500 PM) for 30 min. The plate-

lets were then collected by centrifugation and resuspended in

fresh medium containing the same concentration of BW755C

present during the preincubation period. Ionophore A23187 (10

PM) was then added, and the platelet suspensions were in-

cubated with stirring for 30 min at 37°C. At the end of this

experimental incubation period, methanol (1 vol.) containing 5

nCi of 12-[3H,]HETE (to monitor recovery) was added. Plate-

let debris was removed by centrifugation, and the supernatant

was acidified and extracted with CH,CI,. The concentrated extract was then analyzed by RP-HPLC (column I, solvent I)

with continuous flow-through ultraviolet monitoring at 235 nm.

HHT is the cyclooxygenase product 12-hydroxyheptade-

catrienoic acid. 12-HETE is the 12-lipoxygenase product 12-hy-

droxyeicosatetraenoic acid. Both compounds contain a con-

jugated diene chromophore and therefore absorb at 235 nm.

Similar tracings were obtained on each of five occasions.

islet (0.02 < P < 0.05). In the presence of 20 PM

ETYA, the increment in secretion at 28 mM glu-

cose vs. 3 mM glucose was greater in the presence of 5 PM 12-HETE than that in the absence of

12-HETE by 0.77 + 0.19 pU/min per islet (0.02 <

P < 0.05). In the presence of 20 PM ETYA, the

increment in secretion at 28 mM vs. 3 mM glucose

as a percent of the control (no ETYA) increment

was also greater in the presence of 5 PM 1ZHETE

than in the absence of IZHETE by 14 f 2.9%

(0.01 < P < 0.02). Although statistically signifi-

cant, the effect of 5 PM IZHETE to reverse the

suppression of glucose-induced insulin secretion by ETYA was thus rather small. At a concentra-

tion of 50 PM, 1ZHETE itself suppressed glucose-induced insulin secretion (Table II), as did

all other tested fatty acid derivatives at this con-

centration.

The fact that 12-lipoxygenase products failed to

* Islet insulin secrehon

0 Mel IL-HETE synthesis

0 Platelet I2-HETE synthesis

h

0 20 100 200 500

BW755C CONCENTRATION (uM)

Fig. 3. Comparison of the concentration dependence of the

inhibition of glucose-induced insulin secretion and of 12-HETE

synthesis by BW755C. Glucose-induced insulin secretion (0)

from isolated pancreatic islets in the presence of various con-

centrations of BW755C (O-500 PM) was determined as de-

scribed in Table 1. The parameter (0) plotted is the rate of

insulin secretion at 28 mM glucose minus that at 3 mM glucose

at a given concentrationof BW755C (‘experimental increment’)

divided by the ‘control increment’ (rate of insulin secretion at

28 mM glucose minus that at 3 mM glucose in the absence of BW755C). Each point (@) represents the mean of four experi-

ments. Standard errors of the mean (n = 4) are indicated. Rat

platelets 12-HETE synthesis (0) was determined as described

in Fig. 2. Each point (0) represents the mean of five experi-

ments. Synthesis of 12-HETE by isolated pancreatic islets (0)

was determined as described in Fig. 1. Each point (0) repre-

sents the mean of two experiments.

32

TABLE III

CONCENTRATION DEPENDENCE OF INHIBITION OF

GLUCOSE-INDUCED INSULIN SECRETION FROM ISO-

LATED PANCREATIC ISLETS BY NORDIHYDRO-

GUAIARETIC ACID (NDGA)

Conditions were as described in Table I except that the inhibi-

tor was nordihydroguaiaretic acid (NDGA) rather than ETYA,

and no fatty acid derivatives were tested as potential secreta-

gogues.

Glucose

concentration

(mM)

3

28

3

28

Inhibitor

None

None

NDGA

(5 PM) NDGA

(5 @It

Insulin

secretion

(&J/mm per islet)

1.35i0.28

5.38 f 1.75

0.98+0.19

5.23 i 0.78

3 NDGA 1.10+0.32

(15 PM) 28 NDGA 3.92 * 0.64

(15 aM)

3 NDGA 0.89 f 0.28

(50 PM) 28 NDGA 1.34 f 0.21

(50 BM)

reverse completely the suppression of glucose-in-

duced insulin secretion by ETYA suggested that

some action of ETYA other than inhibition of the

12-lipoxygenase might be responsible for suppres-

sion of insulin secretion. If that were the case,

inhibitors of the 1Zlipoxygenase structurally dis- similar to ETYA might not inhibit insulin secretion or might do so only at concentrations much

higher than those required for inhibition of the

12-lipoxygenase. The compound BW755C has been reported to inhibit the cyclooxygenase and the IZlipoxygenase in human platelets [3.5,36] and was found also to do so in rat platelets (Fig. 2) although higher concentrations of the agent were required with rat platelets. BW755C also inhibited glucose-induced insulin secretion and rat islet synthesis of 12-HETE with similar concentration

dependence (Fig. 3). The compound nordihydroguaiaretic acid

(NDGA) inhibits the human cyclooxygenase and 12-lipoxygenase [35,36] and was also found to

inhibit glucose-induced insulin secretion (Table III)

with 50% inhibition occurring at a concentration

of about 25 PM. Rat islet synthesis of I2-HETE

was inhibited by 38% at 15 PM NDGA and by

96% at 50 yM NDGA as determined by stable-iso-

tope dilution GC-MS measurements. Although the compound esculetin has been reported to inhibit lipoxygenases under some circumstances

[37,383, neither glucose-induced insulin secretion nor islet 12-HETE synthesis was inhibited by

esculetin at concentrations up to 200 FM (data not

shown).

One explanation of the observations that glucose-induced insulin secretion is suppressed by

three structurally distinct lipoxygenase inhibitors

and yet uninfluenced by exogenous 12-HPETE is

that synthesis of products by an islet lipoxygenase

other than the 1Zlipoxygenase participates in glucose-induced insulin secretion. Although 12-HETE

TABLE IV

INFLUENCE OF EXOGENOUS 15-HYDROXY~ICOSA-

TETRAENOIC ACID (15-HETE) AND OF 15 HYDROPER-

OXYEICOSATETRAENOIC ACID (IS-HPETE) ON IN-

SULIN SECRETION FROM ISOLATED PANCREATIC

ISLETS

Condition were as described in Table I except that either

15-hydroperoxyeico~tetraenoic acid (IS-HPETE) or 15hy-

droxyeicosatetraenoic acid (IS-HETE) (5 &M) rather than 12-

HPETE was tested as a potential secretagogue, and the inhibi-

tor was BW755C rather than ETYA. When inhibitor was

present it was 500 PM.



(mM) f $J/min per islet)

3 None None 0.46 f 0.02

28 None None 4.60 & 0.05

3 BW755C None 0.49f0.12

28 BW755C None I.36kO.19

3 None IS-HETE 0.56 t 0.25 28 None 15HETE 3.82kO.67

3 BW755C 15-HETE 0.39 * 0.09

28 BWl55C 15-HETE I .74 & 0.23

3 None 15-HPETE 0.47 * 0.01

28 None IS-HPETE 4.61 5 1 .a5

3 BW755C 1%HPETE 0.55 io.12 28 BW755C IS-HPETE 1.62&O&l

is the most abundant lipoxygenase product from

islets, trace amounts of 15-HETE are produced. The effects of exogenous 15-HPETE and 15-HETE on insulin secretion were therefore determined. At a concentration of 5 FM, neither compound in-

fluenced basal or glucose-stimulated secretion and neither reversed the inhibition of glucose-stimu-

lated secretion by BW755C (Table IV). At a concentration of 50 PM, both compounds inhibited

glucose-induced insulin secretion by about 30%

(data not shown). Although no 5-HETE was detected from iso-

lated islets, it was considered possible that the

precursor 5-HPETE might be converted to prod-

ucts other than 5-HETE [39-421 or that 5-HETE might be incorporated into membrane phospholi-

pid [43&t] and thereby influence insulin secretion.

The effects of exogenous 5-HPETE and 5-HETE on insulin secretion were therefore determined. At

a concentration of 5 PM, neither compound reversed the suppression of glucose-induced insulin

secretion by BW755C (Table V) or ETYA (data

not shown), and neither compound influenced insulin secretion at 3 or 28 mM glucose (Table V).

At a concentration of 50 PM, both 5-HETE and

5-HPETE suppressed glucose-induced insulin

TABLE VI

33

TABLE V

INFLUENCE OF EXOGENOUS 5-HYDROXYEICOSA-

TETRAENOIC ACID (S-HETE) AND OF 5-HYDROPER-

OXYEICOSATETRAENOIC ACID (5-HPETE) ON IN-

SULIN SECRETION FROM ISOLATED PANCREATIC

ISLETS

Conditions were as described in Table I except that either

5hydroperoxyeicosatetraenoic acid (5-HPETE) or 5-hydroxy-

eicosatetraenoic acid (5HETE) (5 PM) rather than 12-HPETE

was tested as a potential secretagogue, and the inhibitor was

BW755C (500 PM) rather than ETYA.




3 None None 0.76 f 0.10

28 None None 4.57k0.16

3 BW755C None 0.71 f 0.08

28 BW755C None 1.93kO.12

3 None 5-HPETE 0.73 + 0.07

28 None 5-HPETE 4.75 f 0.15

3 BW755C 5-HPETE 0.71 f 0.14

28 BW755C 5-HPETE 1.96 f 0.26

3 None 5-HETE 0.63 f 0.07

28 None 5-HETE 4.77 f 0.28

3 BW755C 5-HETE 0.78rtO.11

28 BW755C 5-HETE 1.77k0.12

INFLUENCE OF EXOGENOUS ARACHIDONIC ACID ON INSULIN SECRETION FROM ISOLATED PANCREATIC

ISLETS IN THE PRESENCE AND ABSENCE OF INHIBITORS OF ARACHIDONATE METABOLISM

Conditions were as described as in Table I except that the inhibitor was either indomethacin or BW755C rather than ETYA, and the

potential secretagogue tested was arachidonic acid (20 PM) rather than 12-HPETE. Statistical comparisons were performed with

Student’s r-test (two-tailed). n.a. denotes not applicable.

Glucose

concentration

(mM)

3

28

3

28

3

28

3

28

3

28

3

28

Inhibitor

None

None

Indomethacin

(lo CM)

BW755C

(509 PM)

None None

lndomethacin

(lo CM)

BW755C

(509 PM)

Potential

secretagogue

None

None

None

None

None

None

Arachidonate

(20 BM)

Arachidonate

(20 c M)

Arachidonate

(20 IBM)

Insulin Significance secretion of difference

($J/min per islet) from control

0.74 f 0.06 n.a. 3.56 + 0.47 n.a

0.66 + 0.07 0.500~P<1.ooo 3.67 f 0.66 0.500 c P < l.ooo

0.86kO.12 0.200 < P < 0.500 1.00~0.40 0.010 i P c 0.020

1.31 f0.16 0.020 < P < 0.050 3.05 f 0.44 .0.200 < P < 0.500

1.33 f 0.32 0.100 < P < 0.200 2.78 rt0.24 0.200 < p < 0.500

1.57kO.16 0.005 < P -z 0.010 1.71 f 0.29 0.010 -z P < 0.020

34

secretion by about 30% (data not shown).

Arachidonic acid itself (20 PM) was found to

increase insulin secretion slightly (1 .&fold) but

significantly (P -c 0.05) with 3 mM glucose but not

with 28 mM glucose (Table VI). This stimulatory

effect of arachidonic acid on insulin secretion with

3 mM glucose did not appear to be attributable to

the generation of arachidonate cyclooxygenase or

lipoxygenase products, since the response was not

significantly influenced by indomethacin or

BW755C (Table VI).

Discussion

The findings in this study indicate that pancreatic islets isolated from adult rats synthesiz,: the

arachidonate 12-lipoxygenase product, I2-HETE,

in amounts of 1.7-2.8 ng per lo3 islets. No detec-

table amounts of 5-HETE and only trace amounts

of I5-HETE were obtained from the islets. The

compounds BW755C and NDGA were shown to

inhibit the synthesis of 12-HETE from endogenous

arachidonate by rat islets and to suppress

glucose-induced insulin secretion with a similar

concentration dependence. As previously reported

[2], the lipoxygenase inhibitor ETYA also sup-

pressed glucose-induced insulin secretion. This suppression was not reversed by exogenous addi-

tion of the 12-lipoxygenase product 12-HPETE and was only partially reversed by its reduction

product, 12-HETE. These synthetic compounds

were highly purified, carefully monitored for sta-

bility and demonstrated to be in solution by ultra-

violet spectrometry. Exogenous 15-HPETE, 5- HPETE, 15-HETE and 5-HETE also failed to

reverse the suppression of glucose-induced insulin

secretion by lipoxygenase inhibitors. The lack of

effect of these exogenous lipoxygenase products is

difficult to attribute to an impaired stimulus-secre-

tion mechanism in the isolated islet preparations because in each experiment in which the lipoxygenase products were tested we observed stimulation of insulin secretion with 28 vs. 3 mM glucose and suppression of the stimulation with the lipoxygenase inhibitors.

These observations argue against the recently suggested hypothesis that the 5-lipoxygenase product, 5-HETE, modulates glucose-induced insulin from isolated islets (91, since we have been unable

to demonstrate a insulin secretagogue effect of exogenous 5-HETE and have been unable to demonstrate islet synthesis of 5-HETE from endoge-

nous arachidonate. Other 5-lipoxygenase products.

such as leukotriene B4, have been reported to stimulate insulin secretion from perfused pancreas

preparations [12]. although not from monolayer

cultures of pancreatic cells from neonatal rats [7].

We have previously been unable to demonstrate synthesis of the 5-lipoxygenase products

leukotriene B4 and 5-HETE from radiolabelled

arachidonate by isolated pancreatic islets [l], and

we observe no insulin secretagogue effect for 5-

HPETE, the precursor of leukotriene B, and 5-

HETE. Others have reported synthesis by isolated

islets of 5-HETE from radiolabelled arachidonate, but this metabolite was characterized only by RP-

HPLC [9] mobility. We have observed material with RP-HPLC mobility similar to 5-[3H]HETE

from isolated islets incubated with [‘Hlarach-

idonate [1,2], but this material is distinguishable

from 5-HETE upon subsequent NP-HPLC analy-

sis [1,2]. In the present report, quantitation of islet

production of HETE isomers from endogenous arachidonate by HPLC analysis followed by sensi-

tive and specific stable isotope dilution gas chro- matographic-mass spectrometric measurements

revealed no detectable amount of 5-HETE, despite the clear demonstration of the synthesis of sub-

stantial amounts of 12-HETE.

Our inability to demonstrate an insulin

secretagogue effect of exogenous 12-HPETE on

isolated pancreatic islets is in contrast to the re-

ported ability of 12-HPETE to stimulate insulin secretion from monolayer cultures of pancreatic

cells derived from neonatal rats (6,7]. This may reflect different roles for metabolites of arachidonic acid in insulin secretion in the two systems. This

possibility is also suggested by the observations that cyclooxygenase inhibitors enhance insulin secretion by the monolayer cultures [45] but not by isolated islets [2,9] and that phospholipase inhibitors suppress insulin secretion by isolated islets (81 but not by the monolayer cultures [46]. In addition, exogenous 12-HPETE is reported to produce only about a 30% increment in insulin secretion from the monolayer cultures of pancreatic cells from neonatal rats [6,7]. This increment is quite small compared to the 400-1000% increases in

35

insulin secretion obtained with maximally stimulatory concentrations of glucose from isolated islets [2], although the insulin secretory response to glucose by the cultured neonatal cells is somewhat smaller [4,6]. It is therefore not clear that the magnitude of the insulin secretory response to 1ZHPETE is sufficient to account for the magnitude of the suppression of glucose-induced insulin secretion by the lipoxygenase inhibitors ETYA, NDGA and BW755C. No lipoxygenase product yet tested has been demonstrated by us or by other investigators [6,7,9,12] to reverse the suppression of glucose-induced insulin secretion by lipoxygenase inhibitors to a substantial degree. The possibility that BW755C, NDGA and ETYA might interfere with the action of lipoxygenase products as well as with their synthesis cannot, however, be excluded.

Several observations have suggested the hypothesis that a 12-lipoxygenase product might participate in glucose-induced insulin secretion from isolated islets: (1) lipoxygenase (but not selective cyclooxygenase) inhibitors suppress glucose-induced insulin secretion from isolated islets [2,9,10]; (2) the 12-lipoxygenase product 12-HETE is the most abundant arachidonate metabolite so far identified from isolated islets [l], and 5-, or 15- lipoxygenase products are not obtained from islets in comparable amounts; (3) concentrations of glucose which stimulate insulin secretion also stimulate the synthesis of 12-HETE by isolated islets [2].

The failure of exogenous 1ZHPETE to reverse the suppression of glucose-induced insulin secretion by lipoxygenase inhibitors does not support the hypothesis that a 12-lipoxygenase product participates in glucose-induced insulin secretion but does not exclude it. Exogenous 12-HPETE may be reduced or otherwise transformed before reaching some critical intracellular target or before under- going conversion to a more potent pro-secretory product such as 8-hydroxy-11,12-epoxy-eicosa- 5,9,14-trienoic acid [47]. Insulin secretion may also require a sequence of events (including 1ZHPETE generation) which is ordered in time and which is not mimicked by sudden addition of large concentrations of exogenous 1ZHPETE at the time of stimulus initiation. It is also possible that arachidonate metabolites in addition to 12-HPETE are required for glucose-induced insulin secretion

and that the addition of any single metabolite is insufficient to promote secretion. Glucose stimu- lates the production of prostaglandin E,, prostaglandin FZol and thromboxane B, as well as 12- HETE by isolated islets [2]. One or more of these cyclooxygenase products may cooperate with 12- HPETE and other mediators in the initiation or maintenance of the secretory process. The lipoxygenase inhibitors ETYA, NDGA and BW755C inhibit the cyclooxygenase as well as the lZlipo- xygenase [1,2,35,36], and inhibition of both pathways may be required for suppression of glucose-induced insulin secretion. Another possibility is that islet paracrine effects might moderate direct effects of 12-HPETE on the beta cell if, for exam- ple, IZHPETE provoked somatostatin release from islet endocrine cells. There is some evidence that arachidonate metabolites may participate in somatostatin secretion in other tissues [48].

The failure of exogenous 1ZHPETE to reverse the suppression of glucose-induced secretion by lipoxygenase inhibitors also could indicate that the suppression of secretion is not due to inhibition of the 1Zlipoxygenase but rather to inhibition of some other process, such as cytochrome P-450- mediated oxygenation of arachidonate. Trienoic expoxides derived from arachidonate via the action of a cytochrome P-450 enzyme have been reported to stimulate insulin secretion from isolated islets, and lipoxygenase inhibitors have been reported to inhibit the oxygenation of arachidonate by cytochrome P-450 from other tissues [lo]. It is not known whether islets are capable of oxygenat- ing arachidonate via cytochrome P-450, but reported attempts at demonstrating islet synthesis of such metabolites have been unsuccessful [lo]. Closer scrutiny of this question will require the preparation of much larger quantities of islets than those so far tested [lo] and the synthesis of suita- bly labelled standards of the cytochrome P-450 products.

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

36

Washington University Biomedical Research Sup-

port Grants Program (NIH BRSG SO7 RR053&9), the Pharmaceutical Manufacturer’s Association

Foundation, the Juvenile Diabetes Foundation and

the National Institutes of Health (AM 34388). The

excellent technical assistance of Richard Thoma,

Deidre Buscetto and C. Joan Fink has been greatly

appreciated as has the interest and advice of Dr.

Jay McDonald, Dr. Paul Lacy an Dr. Philip

Needleman. Special thanks are due to Jane Huth

for the preparation of the manuscript. We are

grateful to Dr. Aubrey Morrison and to Dr. Wil-

liam Sherman for providing access to the Hewlett- Packard 5985B mass spectometer within the

Washington University Mass Spectrometry Re-

source Center which is supported by NIH grasrt

RR 00954.

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Arachidonic acid metabolism in isolated pancreatic islets. III. Effects of exogenous lipoxygenase...

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