RostagiamJins and Essential Fatty Acids (1990) 40.214 @ Longman Group UK Ltd 1990

Lipoxygenase-Generated Icosanoids Inhibit Glucose-induced Insulin Release from Rat Islets

M. H. Nathan* and S. Belbez Pekt *Department of Internal Medicine (Division of Endocrinology and Metabolism), University of Michigan, Ann Arbor, Michigan, USA and ‘5560 Medical Sciences Research Building-2, University of Michigan Medical Center, Ann Arbor, MI 48109-0678, USA (Correspondence to SBP)

ABSTRACT. -pathway metabolites of are produced in pancreatic islets. They are are implicated in insulin release, since nonselective inhibitors of inhibit glucose-induced insulin release. We studied the interplay in insulin release between glucose and selected icosanoids formed in 5-, 12- and 15-lipoxygenase pathways. Effects on immunoreactive insulin release of 10’ to 1O-6 12-(R)-HETE, 12-(S)-HETE, hepoxilin As, Bq, LTB4 or LTC4 were tested individually in 30-min incubations of freshly isolated young adult Wistar rat pancreatic islets, in the presence of 5.6 mM or 23 mM glucose. Basal insulin release (at 5.6 mM glucose) was stimulated by LTC4 and hepoxilin A3 (304% and 234% of controls at 5.6 mM glucose alone, respectively), inhibited by 12-(S)-HPETE (56%), and was not affected by 12-(R)-HETE, 12-(S)-HETE, lipoxin B4 or LTB4 (ill%, 105%, 106% and 136%, respectively). Insulin release evoked by 23 mM glucose (190-320%) was inhibited (50-145%) by all icosanoids tested, except LTC4 (162%). We conclude that, among the lipoxygenase products tested, only leukotrienes and hepoxilin are candidates for a tonic-stimulatory influence on basal insulin release. Since glucose promotes icosanoid formation in islets, the observed inhibition of glucose-induced insulin release by lipoxygenase products suggests the existence of a negative-feedback system.

INTRODUCTION evoked by a lipoxygenase inhibitor, icosatetraynoic acid (ETYA), was reversed partially by the addition In many mammalian tissues, 5-, 12- and 15- of 12-HETE. Among the hydroperoxy- lipoxygenases exist, which convert arachidonic acid icosatetraenoic acids (HPETEs), 12-HPETE and (5,8,11,14-icosatetraenoic acid) into hydroperoxy- 15-HPETE stimulated insulin release in neonatal, and hydroxy-icosatetraenoic acids, leukotrienes, but not in adult rat islets (8). Yamamoto et al (7) hepoxilins and . Icosanoids formed in the found that 15-HPETE inhibited glucose-induced in- lipoxygenase pathways are of potential interest sulin release. An epoxide formed in the in regards to pancreatic islet function, in that non- 12-lipoxygenase pathway, 8-hydroxy-11, 12-epoxy- selective inhibitors of lipoxygenases consistently icosatrienoic acid (hepoxilin A3) stimulated insulin attenuate stimulated insulin release in vitro (l-6). release in the presence of 10 mM glucose (9). We In search for the identity of the putative insulin reported that 5, 12-dihydroxy-icosatetraenoic acid secretagogue, individual exogenous lipoxygenase- ( Bq, LTBJ,5-hydroxy-6-S-glutathionyl- pathway products have been administered to the icosatetraenoic acid (LTCJ, and.to a lesser extent pancreas or pancreatic islets. Among the hydroxy- 5-hydroxy-6-S-cysteinylglycinyl- icosateraenoic acid icosatetraenoic acids (HETEs), Yamamoto et al (7) (LTD4) and 5-hydroxy-6-S-cysteinyl- icosatetraenoic found that 5-HETE stimulated basal insulin release; acid (LTE,J stimulated insulin secretion from the on the other hand, 12-HETE and 15-HETE in- isolated, perfused rat pancreas (10). Metz et al (4) hibited glucose-induced insulin release. Metz et al found that an epoxy-derivative of LTA4 stimulated (4) did not observe any effect of 1ZHETE in neo- insulin release from neonatal rat islets. natal rat islets; yet, Turk et al (1) reported that Evidence has been sought for the biosynthesis of the attenuation of glucose-induced insulin release lipoxygenase-pathway products of arachidonate in pancreatic islets. A major icosanoid formed from

Date received 20 September 1989 arachidonate in islets is 12-HETE (11, 12). Pace-As- Date accepted 20 November 1989 ciak and Martin (9) reported that rat islets produce

21 22 Prostaglandins Leukotrienes and Essential Fatty Acids

hepoxilin As. We provided preliminary evidence for washed with Krebs-Ringer-Hepes buffer (KRH), the production of leukotrienes (13); although Turk and preincubated in 200 p KRH containing 5.6 mM et al (12, 14) failed to confirm this finding. glucose and 0.1% bovine serum albumin, A potential interplay between the lipoxygenase for 60 min at 37°C in an atmosphere of 95% O2 pathway products and glucose in islets is suggested. and 5% COZ. Upon completion of the preincuba- First, an increase in ambient glucose concentration tion, icosanoids or their vehicle (100% ethanol) ap- leads to increased production of these icosanoids in propriately diluted in a volume of 400/~1 and islets (11, 15). Second, the attenuation of insulin 400 ~1 of buffer containing glucose to yield a final secretion with inhibitors of lipoxygenases has been glucose concentration of either 5.6 mM or 23 mM demonstrated mostly with glucose-induced insulin were added to each test tube. The final concentra- release. The present study represents a unifying ef- tions of the icosanoids were: 12-(R)-HETE or fort to establish which icosanoids formed in the 12-(S)-HETE 10-M; 12-(S)-HPETE 10%; LTB4 l@ three lipoxygenase pathways are secretagogues of 6M; LTC4 6x 10M7M; hepoxilin A3 lo-6M; and insulin, and whether their actions are influenced by lipoxin B4 M. The l.O-ml samples were incubated glucose in isolated rat islets. Our results indicate for 30 min at 37°C. Thereafter, the tubes were that LTC4 and hepoxilin A3 stimulate insulin release chilled rapidly and centrifuged at 800 g for 3 min at a “basal” concentration of glucose, and, with the at 4°C. The supernatants were removed and stored exception of LTC4, all lipoxygenase products tested at-20°C for subsequent measurement of the levels of inhibit glucose-induced insulin release. insulin. The levels of insulin in the incubation media were measured by a ‘double-antibody’ radioim- MATERIALS AND METHODS munoassay, in which a rat insulin antiserum and rat insulin standards were employed (17). The inter- The supplier of collagenase (type V) and Ficoll was assay variability was 6-9%. Sigma, St. Louis, MO, and that of 12-(R)-HETE 12- At least three experiments of each kind were car- (S)-HETE, 12-(S)-HPETE, LTB4, LTC4, hepoxilin ried out, each time using a different batch of islets. As as methyl ester, and 5, 6, 15trihydroxy- Each experimental condition was run in triplicate. icosatetraenoic acid (lipoxin BJ as methyl ester On each sample, the radioimmunoassay was carried Cayman Chemical, Ann Arbor, MI. All icosanoids out in duplicate, and the mean result of the repli- were shipped in dry ice in ethanol and were stored cates was computed. To accommodate the variation at -70°C. in insulin-secretory capacity among different batches On the day of each experiment, pancreatic islets of islets, the data for each experiment were normal- were isolated from 250 to 350-g male Wistar rats, ized by setting the result obtained with 5.6 mM which had free access to food and water until the glucose alone to 100%. The means from all experi- time of the procedure. The method of Gotoh et al ments of the same kind were used to obtain the (16) was used, in which the digestion of the mean &SEM for that test condition, The statistical pancreas proceeded without shaking, followed by a significance of the differences among means was density gradient and manual sorting of the islets. determined by analysis of variance using Dunnett’s Ten islets were transferred into each test tube, tetesting.

Table Immunoreactive insulin release from isolated rat islets in response to icosanoids formed in various lipoxygenase pathways, in the presence of low and high concentrations of glucose

Icosanoid (N) Insulin Release (Mean + SEM Percent)* With 5.6 mM Glucose With 23 mM Glucose Icosanoid No Icosanoid Icosanoid

LTB,; 1U6 M (3) 136 + 12 250 + 51t 130 * 19t LTC,; 6 x 1(r7 M 304 + 32t 250 f 51t 162 + 15 12-(S)-HPETE; 10” M ;; 56 + 6-t 139 + 11t 50 f St 12-(R)-HETE; 10” M 111 f 19 190 + lot 91 + 14t 12-(S)-HETE; 10” M 104.5 + 6.6 190 2 lot 112 i 187 Hepoxilin A,; lo” M tz; 234 + 39t 320 f 33t 145 + 287 Lipoxin B,; 10” M (3) 106.4 + 12 320 + 33t 101 + 27t Footnotes: The final concentration of each icosanoid is given in parentheses next to its name. For each experimental condition, the number of observations is given in parentheses. [*] The data for each experiment were normalized by setting the result obtained with 5.6 mM glucose alone to 100%. [t] p

RESULTS 1Zlipoxygenase product hepoxilin As stimulated the basal secretion of insulin. In this regard, LTC4 was When the data from all experiments were compiled, by far the more potent one, despite the fact that it over the 30-min incubation period, in the absence was employed at a concentration lower than all of any icosanoids, the secretion of insulin was 229 other icosanoids. We reported previously that, in +40&‘islet/h with 5.6 mM glucose and 410 f 55 the perfused rat pancreas, in the presence of &islet/h with 23 mM glucose (N=13; ~~0.01). 5.6 mM glucose, LTB4 as well as LTCa stimulate in- The data on the effects of individual icosanoids at sulin secretion at similar potencies in a dose-related the two concentrations of glucose are given in the manner in the concentration range of lo-” M to 10e7 Table. M (10). In the present study, the mild stimulatory The effects of two 5-lipoxygenase pathway effect of LTB4, at a concentration higher than those products were tested. Under basal conditions employed previously, did not attain statistical sig- (5.6 mM glucose), LTC4 stimulated the release of nificance. In addition to the obvious differences in insulin in the presence of 5.6 mM glucose; LTB4 the experimental conditions, the possibility of had a mild stimulatory effect which did not attain tachyphylaxis may be considered. Our observation statistical significance. The stimulatory effect of of an agonist action of hepoxilin As in the presence LTC, exceeded that of LTB4 (304% KS 136%, p of 5.6 mM glucose is an extension of that of Pace- ~0.05). LTC4 did not modify the release in Asciak and Martin (9) that hepoxilin stimulated response to 23 mM glucose; on the other hand, insulin release at 10 mM glucose. LTB, inhibited glucose-induced insulin release. Thus, to this date, among the lipoxygenase- Four 12-lipoxygenase pathway products were pathway products, LTB4 and peptidyl LTs (10, and selected. 12-(S)-HPETE inhibited insulin release in present study), 5-HETE (7); and hepoxilin A3 (9) the presence of 5.6 mM as well as 23 mM glucose. have been shown to stimulate basal insulin secretion Neither 12-(R)-HETE nor 12-(S)-HETE had any ef- on their own. fect on basal insulin release. In the presence of In our study, 12-(S)-HPETE inhibited the basal 23 mM glucose, 12-(R)-HETE as well as 12-(S)- release of insulin. Metz et al (4) reported that 12- HETE inhibited glucose-induced insulin release. HPETE stimulated insulin release in neonatal rat Hepoxilin A3 stimulated insulin secretion in the islets, but had no effect in adult islets. The reasons presence of 5.6 mM glucose, but inhibited insulin for the age difference in the insulin-secretory release evoked by 23 mM glucose. responses observed by Metz, and the contrasting in- Both 5-lipoxygenase and 15-lipoxygenase are in- hibitory response which we observed remain to be volved in the biosynthesis of lipoxin. B4 Lipoxin had clarified; differences in experimental conditions, or no effect on basal insulin secretion. It inhibited in- the fact that we used selectively the (S) enantiomer sulin release stimulated by 23 mM glucose. rather than an unspecified preparation of 12- Statistical comparison of the insulin-secretory HPETE may have affected the outcome. responses to individual lipoxygenase-pathway 1ZHPETE is chemically unstable; conversion of 12- products in the presence of 5.6 mM glucose vs in HPETE to another product in islets may be the presence of 23 mM glucose did not reveal any considered as being responsible for the inhibitory ef- significant differences. fect. 12-HPETE‘ is the immediate precursor of 12-HETE; reportedly, 12-HPETE can be converted also to hepoxilin in islets (9). Our results with ex- ogenous 12-HETEs or hepoxilin do not lend support DISCUSSION to the possibility that these compounds mediated the inhibitory effect of 12-HPETE, in that 12-HETE Our results indicate that icosanoids formed in the had no effect on, and hepoxilin stimulated basal in- lipoxygenase pathways affect insulin secretion in a sulin release. Nevertheless, some other compound complex manner. formed in islets from 1ZHPETE may have been Since 5.6 mM is slightly below the blood level of responsible for the inhibition. glucose which prevails in the fasting rat, and since Neither the (R) nor the (S) enantiomer of 12- the islets had been exposed to this concentration HETE stimulated the basal secretion of insulin. In during the 60-min preincubation followed by the 30- previous studies, 12-HETE preparations of un- min incubation periods, we assumed that the insulin known type had been used. We had considered that secreted with 5.6 mM glucose represented the stereospecificity may be a determinant in any ‘basal’ secretion, and interpreted the response to secretagogue action; for example, the (R) en- an icosanoid at this concentration of glucose as an antiomer of 1ZHETE may interact with LTB4 effect of that icosanoid on its own. receptors (18). Still, the employment of the in- Among the seven icosanoids representing the dividual enantiomers of 12-HETE failed to uncover products formed in the three lipoxygenase path- an agonist action which previously may have been ways, the 5-lipoxygenase product LTC, and the overlooked. 24 Prostaglandins Leukotrienes and Essential Fatty Acids

The effect of lipoxins on the secretion process lipoxygenases. The fact that chemically unrelated previously had not been tested. Lipoxin B4 failed to lipoxygenase inhibitors have the same effect affect basal insulin secretion. In the case of lipoxin, weakens this possibility. Another possibility is that as well as in the case of the two enantiomers of 12- the inhibition of lipoxygenases may lead to in- HETE, one may consider that they were inhibitory creased production in alternate pathways of certain in the basal state, but that the model employed was arachidonate metabolites that could be inhibitors of not sufficiently sensitive to demonstrate the inhibi- stimulated insulin release. A third possibility is that tion. This is unlikely, since the inhibitory action of other icosanoids formed in islets in the lipoxygenase 12-(S)-HPETE could be revealed. pathways, the identity of which is obscure at this The striking novel finding in our study is that, time, may have tonic-stimulatory properties without with the exception of LTQ, all lipoxygenase path- interfering with the glucose signal, the formation of way products employed exerted an inhibitory action which is inhibited by the lipoxygenase inhibitors. on insulin release, when insulin release had been We conclude that, among the lipoxygenase augmented in the presence of 23 mM glucose. In products tested, only leukotrienes and hepoxilin are most cases, these icosanoids suppressed insulin candidates for a tonic-stimulatory influence on basal release toward or to, but not below the levels ob- insulin release. Our observation that lipoxygenase served in the presence of 5.6 mM glucose alone. products inhibited glucose-induced insulin release, Hence, we believe that the lipoxygenase products together with the existing evidence that glucose aug- interfered with the stimulatory signal for insulin ments icosanoid formation in pancreatic islets, secretion elicited by glucose, irrespective of suggest the existence in islets of a negative-feedback whether, on their own, they may have a stimulatory system involving these icosanoids, or inhibitory effect, or no effect on insulin release. An extrapolation of our findings with exogenous lipoxygenase-pathway products to their endogenous Acknowledgements counterparts would assign to the latter a negative rather than a positive modulatory role in glucose- The study was supported in part by the United States Public Health Service grants ROl DK21192, T32 DK07245, F32 induced insulin release. Since glucose promotes the DK08282 and P60 DK20572 from the National Institute of biosynthesis of icosanoids in islets, including the Diabetes. and Digestive and Kidney Diseases. lipoxygenase pathway products, the intriguing possi- bility emerges that, by inducing the increased References availability of these compounds, glucose engages a restraining system on insulin release. Turk J, Colca J R, McDaniel M L. Arachidonic acid metabolism in isolated pancreatic islets. III. LTC4 stimulated basal insulin release and had no Effects of exogenous lipoxygenase products and effect on glucose-induced insulin release. In the case inhibitors on insulin secretion. Biochim Biophys of hepoxilin, the stimulation of basal, but inhibition Acta 834: 23-36, 1985. Turk J, Wolf B A, McDaniel M L. The role of of glucose-induced insulin release are seemingly phospholipid-derived mediators including paradoxical. These icosanoids may have two or arachidonic acid, its metabolites, and more mechanisms of action in the islet B-cells. One inositoltriphosphate and of intracellular Ca2+ in glucose-induced insulin secretion by pancreatic mechanism may provide for a tonic-stimulatory in- Glets. Progr Lipid Res 26: 125-18i, i987. fluence, while another may interfere selectively with Morean R. Pek S B. Role of arachidonate the signal for insulin release generated by glucose. lipoiygena$e and cyclooxygenase products in insulin and glucagon secretion from rat pancreatic islets. The latter mechanism may have mitigated the Metabolism 33: 928-935, 1984. strong agonist action LTC4 only partially, while it 4. Metz S, Van Rollins M, Strife R, Fujimoto W, may have overwhelmed the action of the weaker Robertson R P. Lipoxygenase pathway in islet endocrine cells: Oxidative metabolism of agonist hepoxilin. In this regard, the lipoxygenase arachidonic acid promotes insulin release. J Clin products are not unique. The promotion of the Invest 71: 1191-1205,1983. basal secretion of insulin by prostaglandins has been Walsh M F, Pek S B. Possible role of endogenous arachidonic acid metabolites in stimulated release observed repeatedly (19-21); yet, others reported of insulin and glucagon from the isolated rat that the same icosanoids inhibited glucose-induced pancreas. Diabetes 33: 929-936, 1984. insulin release (20, 22). Walsh M F, Pek S B. Effects of lipoxygenase and cvclooxveenase inhibitors on glucose-stimulated The results of our study, as well as those of pre- i&lin &retion from the isorated perfused rat vious work by others, do not proyide an explanation pancreas. Life Sci 34: 1699-1706, 1984. for the consistent observation that nonselective in- Yamamoto S, Ishii M, Nakadate T, Nakaki T, Kato R. Modulation of insulin secretion by hibitors of lipoxygenases inhibit stimulated insulin Iipoxygenase products of arachidonic acid. J Biol release. All drugs are likely to have actions in ad- Chem 258: 12149-12152, 1983. dition to the main action attributed to them; hence, Metz S A, Murphy R C, Fujimoto W Y 1984. Effects on rducose-induced insulin secretion of these compounds may be inhibiting insulin release lipoxygena&derived metabolites of arachidonic by mechanisms unrelated to the inhibition of acid. Diabetes 33: 119-124. Lipoxygenase Products and Insulin Release 25

9. Pace-Asciak C R, Martin J M. Hepoxilin, a new metabolic synthesis. Biochim Biophys Acta family of insulin secretagogues formed by intact rat 794: 125-136,1984. pancreatic islets. Prostaglandins Leukotrienes Med 16. Gotoh M. Maki T, Kiyoizumi T, Satomi S, 16: 173-180,1984. Monaco A P. An improved method for isolation of 10. Pek S B, Walsh M F. Leukotrienes stimulate mouse pancreatic islets. Transplantation insulin release from the rat pancreas. Proc Nat1 40: 437-438,1985. Acad Sci USA 81: 2199-2202. 1984. 17. Pek S. Glucagon and insulin. pp 122-140 in 11. Metz S. Glucose increases the synthesis of Nuclear Medicine: Endocrinologv. (Rothfeld B ed) lipoxygenase-mediated metabolites of arachidonic Lippincott. Philadelphia, 1978. -. acid in intact rat islets. Proc Nat1 Acad Sci USA 18. Evans J F, Leblanc Y, Fitzsimmons B J, Charleson 82: 198-202, 1985. S. Nathaniel D, Leveille C. Act’vation of leukocyte 12. Turk J, Colca J R, Kotagal N, McDaniel M L. movement and displacement of L-LTB4 from 1984. Arachidonic acid metabolism in isolated leukocyte membrane preparations by 12(R)- and pancreatic islets. 1. Identification and quantitation 12(S)-hydroxveicosatetraenoic acid. Biochim of lipoxygenase and cyclooxygenase products. Biophys Acta 917: 406-410, 1987. Biochim Bioohvs Acta 794: 110-124. 19. Johnson D G, Fujimoto W Y, Williams R H. 13. Pek S B, W&b M F. Effects of icosanoids on in Enhanced release of insulin by prostaglandins in vitro secretion of islet hormones. Excerpta Medica isolated pancreatic islets. Diabetes 22: 658-663, Intl Cong Ser 700: 198-202, 1986. 1973. 14. Turk J, Wolf B A, Comens P G, Colca J, Jakschik 20. Burr I, Sharp R. Effects of prostagtandin E, and B, McDaniel M L. Arachidonic acid metabolism in epinephrine on the dynamics of insulin release in isolated pancreatic islets. IV. Negative ion-mass vitro. Endocrinology 94: 835-839. 1974. spectrometric quantitation of monooxygenase 21. Pek S, Tai T-Y. Elster A. Stimulatory effects of producty synthesis by liver and islets. Biochim prostaglandins E, , Ez and Fglpha on glucagon and Biophys Acta 835: l-17. 1985. insulin release in vitro. Diabetes 27: 801-809. 1978. 15 Turk J, Colca J R. Kotagal N, McDaniel M L. 22. Robertson R P. Arachidonic acid metabolite Arachidonic acid metabolism in isolated pancreatic regulation of insulin secretion. Diabetes Metab Rev islets. II. The effects of glucose and of inhibitors of 2: 261-296.1986. arachidonate metabolism on insulin secretion and