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Diabetes 56:197-203, 2007
DOI: 10.2337/db06-0490
© 2007 by the American Diabetes Association
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The Role of Arachidonic Acid and Its Metabolites in Insulin Secretion From Human Islets of Langerhans

Shanta J. Persaud1, Dany Muller1, Véronique D. Belin1, Isidora Kitsou-Mylona1, Henry Asare-Anane1, Alexandros Papadimitriou1, Chris J. Burns2, Guo Cai Huang3, Stephanie A. Amiel3, and Peter M. Jones1

1 Beta Cell Development and Function Group, Division of Reproductive Health, Endocrinology, and Development, King’s College London, London, U.K
2 Division of Immunology and Endocrinology, National Institute for Biological Standards and Control, South Mimms, Hertfordshire, U.K
3 Division of Gene and Cell Based Therapy, King’s College London, London, U.K

Address correspondence and reprint requests to Dr. S.J. Persaud, 2.9N Hodgkin Building, King’s College London, London SE1 1UL, U.K. E-mail: shanta.persaud{at}kcl.ac.uk

Abbreviations: AACOCF3, arachidonyltrifluoromethyl ketone; baicalein, 5,6,7-trihydroxyflavone; cPLA2, cytosolic phospholipase A2; COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; LOX, lipoxygenase; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; PACOCF3, palmityltrifluoromethylketone; PGE2, prostaglandin E2; phenidone, 1-phenyl-3-pyrazolidinone


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The roles played by arachidonic acid and its cyclooxygenase (COX)-generated and lipoxygenase (LOX)-generated metabolites have been studied using rodent islets and insulin-secreting cell lines, but very little is known about COX and LOX isoform expression and the effects of modulation of arachidonic acid generation and metabolism in human islets. We have used RT-PCR to identify mRNAs for cytosolic phospholipase A2 (cPLA2), COX-1, COX-2, 5-LOX, and 12-LOX in isolated human islets. COX-3 and 15-LOX were not expressed by human islets. Perifusion experiments with human islets indicated that PLA2 inhibition inhibited glucose-stimulated insulin secretion, whereas inhibitors of COX-2 and 12-LOX enzymes enhanced basal insulin secretion and also secretory responses induced by 20 mmol/l glucose or by 50 µmol/l arachidonic acid. Inhibition of COX-1 with 100 µmol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion. These data indicate that the stimulation of insulin secretion from human islets in response to arachidonic acid does not require its metabolism through COX-2 and 5-/12-LOX pathways. The products of COX-2 and LOX activities have been implicated in cytokine-mediated damage of ß-cells, so selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: they would minimize ß-cell dysfunction while maintaining insulin secretion through enhancing endogenous arachidonic acid levels.

Phospholipases A2 (PLA2) constitute a large family of enzymes that hydrolyze the sn-2 position of membrane phospholipids to generate arachidonic acid. Secretory type I (1) and type II (1,2) PLA2 enzymes have been identified in islets, as have the Ca2+-dependent type IV cytosolic PLA2 (cPLA2) (1–3) and the Ca2+-independent type VI isozymes (4), and it is well-established that insulin secretion from pancreatic ß-cells is stimulated by arachidonic acid (5–7). Although arachidonic acid is known to exert direct functional effects in vitro, it is also further metabolized by cyclooxygenase (COX) enzymes to produce prostaglandins (rev. in 8) and lipoxygenases (LOX) to produce hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (rev. in 9). Two COX genes have been cloned: the COX-1 gene codes for COX-1 and the COX-3 splice variant (10), and the COX-2 gene codes for the COX-2 isoform. Depending on the oxygenation site in arachidonic acid, the LOX enzymes are termed 5-, 12-, and 15-LOX.

The roles played by COX and LOX enzymes in ß-cells have not been fully established, but there is good evidence that both COX-2 (11–14) and 12-LOX (15,16) play roles in cytokine-mediated damage of ß-cells. Furthermore, signaling through the 12-LOX pathway is reported to upregulate COX-2 gene expression (17).

Although there are some contradictory reports about the effects of COX and LOX products on insulin secretion, the consensus view is that prostaglandins (particularly prostaglandin E2 [PGE2]) have inhibitory effects, whereas HETEs and leukotrienes are stimulatory (18–22). However, much of the data has been obtained using inhibitors of questionable specificity, and the situation is complicated by the dearth of studies performed using human islets of Langerhans, the variabilities in experimental protocols, the uncertainty of the LOX isoforms expressed by human ß-cells, and the realization that traditionally used COX inhibitors affect COX-1 and COX-2 with different potencies. An elegant commentary (23) highlighted the requirement for a re-examination of the function of COX enzymes and their products in ß-cell function. We have now carried out a systematic examination of COX and LOX expression and function in human islets to identify the importance of unmetabolized arachidonic acid and its LOX and COX products in the insulin secretory response of isolated human islets.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue culture reagents, BSA (fraction V), {gamma}-globulins, EGTA, PBS, arachidonic acid, and PGE2 were from Sigma-Aldrich (Dorset, U.K.). PCR primers were prepared by Dr. Phil Marsh (Molecular Biology Unit, King’s College London). TaqDNA polymerase was from Promega (Southampton, U.K.), Dynabeads Oligo(dT)25 kit was from Dynal (Oslo), MMLV reverse transcriptase was from Invitrogen (Paisley, U.K.), and the QIAquick gel extraction kit was from QIAgen (Crawley, U.K.). Arachidonyltrifluoromethyl ketone (AACOCF3), palmityltrifluoromethylketone (PACOCF3), and 1-phenyl-3-pyrazolidinone (phenidone) were from BIOMOL Research Laboratories (Plymouth Meeting, PA). 5,6,7-Trihydroxyflavone (baicalein), N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS-398), and REV 5901 were from Calbiochem (Nottingham, U.K.). Na[125I] for iodination of insulin and [125I]cyclic AMP were from GE Healthcare (Buckinghamshire, U.K.), and [3H]arachidonic acid was from PerkinElmer (Buckinghamshire, U.K.).

Isolation of human islets of Langerhans.
Human islets were supplied by the King’s College Islet Transplantation Unit (King’s College Hospital, London). Briefly, pancreata from nondiabetic, cadaver organ donors were removed (with permission), and the islets of Langerhans were isolated under aseptic conditions using a novel method for improved islet yield (24). The islets were maintained for up to 48 h at 37°C (95/5% air/CO2) in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS and 100 units/ml penicillin and 0.1 mg/ml streptomycin.

RT-PCR.
Messenger RNA was isolated from hand-picked human islets and from human platelets and leukocytes, as appropriate (provided by Dr. Kalwant Authi and Dr. Janaka Karalliedde, Cardiovascular Division, King’s College London) using the Dynabeads Oligo(dT)25 kit according to the manufacturer’s instructions. The mRNAs were reverse transcribed into cDNA, as previously described (25), and the primer pairs listed in Table 1 were used in RT-PCRs to detect cPLA2; COX-1; COX-2; COX-3; 5-, 12-, and 15-LOX; and PGE2 receptor (EP1, -2, -3, and -4) cDNAs. All reactions were carried out with 1-min denaturation at 95°C, annealing for 1 min at the temperatures indicated in Table 1, and elongation at 74°C for 2 min. PCR products were separated by agarose gel electrophoresis (2% [wt/vol]) and visualized by staining with ethidium bromide (0.5 µg/ml). All products were cut from the gels and spin column purified using a QIAquick gel extraction kit, and identities were confirmed by sequencing on an ABI 377 using fluorescent chain-terminator methods. In some PCRs, template cDNA obtained from single human islet cells (26) was amplified using the COX-2 primer sets listed in Table 1.


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  		TABLE 1 Primer sequences, annealing temperatures, and MgCl2 concentrations for amplification of human cDNAs encoding cPLA2; COX-1, -2, and 3; 5-, 12-, and 15-LOX; and PGE2 receptor isoforms EP1, -2, -3, and -4

 
Insulin secretion.
The time course of insulin release from human islets of Langerhans was assessed using a multichamber perifusion system at 37°C in a temperature-controlled environment (25). Isolated islets were loaded into Millipore chambers containing 1-µm filters and pre-perifused for 1 h with a bicarbonate-buffered physiological salt solution containing 2 mmol/l glucose. Islets were subsequently perifused (0.5 ml/min) with the salt solution supplemented with compounds of interest, as described in RESULTS, and perifusate samples were collected every 2 min. The insulin content of the samples was determined by radioimmunoassay (27).

[3H]arachidonic acid release.
Human islets were labeled with [3H]arachidonic acid (50 µCi/ml) for 18 h and then perifused with measurement of [3H]arachidonic acid efflux, essentially as previously described (28).

Cyclic AMP generation.
Cyclic AMP content of islet pellets was determined by radioimmunoassay (29) after groups of ~200 human islets had been incubated (37°C, 20 min) in the absence or presence of 1 µmol/l PGE2. Insulin secreted into the supernatant was also measured in the same samples (27).

Statistical analysis.
Data are expressed, where appropriate, as means ± SE and were analyzed statistically using Student’s t tests. Differences between treatments were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
cPLA2, COX, and LOX expression in human islets.
The primers listed in Table 1 were used in RT-PCRs to determine the expression of cPLA2, COX-1, COX-2, COX-3, 5-LOX, 12-LOX, and 15-LOX in mRNA obtained from hand-picked isolated human islets. As shown in Fig. 1A, products of the appropriate sizes for cPLA2, COX-1, and COX-2 were amplified, and the identities of purified PCR products were confirmed by DNA sequencing. COX-3 mRNA was not detected in human islets, but the primers did amplify the expected 120-bp product when genomic DNA was used as a template (Fig. 1A). Single-cell RT-PCR experiments using individual isolated human islet cells indicated that COX-2 mRNA expression was localized to ß-cells and some nonendocrine islet cells (non–{alpha}-, non–ß-, and non–{delta}-cells), but the RNA was not amplified from {alpha}-cells (Fig. 1B). Human islets also expressed mRNAs for 5- and 12-LOX isoforms (Fig. 1C). However, a product was not amplified from human islet cDNA using 15-LOX primers, even after 45 cycles, although a product of the appropriate size and sequence was obtained using a human leukocyte cDNA template (Fig. 1C).


Figure 1
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  		FIG. 1. Detection of cPLA2, COX, and LOX isoforms in human islets. A: mRNAs coding for cPLA2, COX-1, and COX-2 were detected in human islets by RT-PCR. COX-3 was not amplified from human islet cDNA, but an appropriate product was obtained when genomic DNA (gDNA) was used as a template. B: Single-cell RT-PCR indicated that mRNA for COX-2 was detectable in human islet ß-cells and in one of three nonendocrine cells (NEC) examined, but not in human islet {alpha}-cells. C: mRNAs for 5- and 12-LOX were detected in human islets by RT-PCR, but a product was not obtained using 15-LOX primers and human islet cDNA template. Products were obtained for all positive control samples (5-LOX and 15-LOX, human leukocytes; 12-LOX, human platelets).

 
Effect of PLA2 inhibition on insulin secretion from human islets.
An increase in glucose concentration from 2 to 20 mmol/l resulted in a biphasic insulin secretory response by human islets, with an initial peak followed by a sustained plateau for the duration of the glucose stimulus (Fig. 2A). Inhibition of PLA2 enzymes with either 100 µmol/l PACOCF3 or AACOCF3 resulted in a significant (P < 0.01) but incomplete inhibition of the glucose-induced insulin secretory response. The inhibitory effect of 100 µmol/l AACOCF3 was greater than that of PACOCF3, causing an 83 ± 18% decrease in the magnitude of glucose-induced insulin secretion from human islets (Fig. 2A). Interestingly, the PLA2 inhibitors stimulated insulin release at basal glucose levels (2 mmol/l), with a transient stimulation in response to PACOCF3 and a sustained response to AACOCF3 (Fig. 2B).


Figure 2
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  		FIG. 2. Effects of PLA2 inhibitors on insulin secretion from human islets. A: Human islets were perifused with physiological salt solution containing 2 mmol/l glucose for the first 10 min and then with 20 mmol/l glucose alone ({blacktriangleup}) or with 20 mmol/l glucose in the presence of 100 µmol/l AACOCF3 ({circ}) or 100 µmol/l PACOCF3 ({blacksquare}). Results are expressed as a percentage of basal insulin secretion at 2 mmol/l glucose (means ± SE, n = 3). B: Human islets were perifused with physiological salt solution containing 2 mmol/l glucose for the first 10 min and then with 100 µmol/l AACOCF3 ({circ}) or 100 µmol/l PACOCF3 ({blacksquare}). Results are expressed as a percentage of basal insulin secretion at 2 mmol/l glucose (means ± SE, n = 3).

 
Effect of inhibition of COX and LOX enzymes on insulin secretion from human islets.
Figure 3A shows that, as expected, 20 mmol/l glucose produced significant increases in insulin secretion from human islets. Elevating arachidonic acid levels during glucose-induced insulin release, either through the addition of exogenous arachidonic acid or through selective inhibition of COX-2 (10 µmol/l NS-398) or 12-LOX (1 µmol/l baicalein), resulted in further increases in insulin secretion. Thus, arachidonic acid, NS-398, and baicalein produced 1.8 ± 0.2-, 2.4 ± 0.6- and 1.7 ± 0.3-fold increases in insulin secretion above the 20 mmol/l glucose-stimulated plateau (means ± SE, n = 3, P < 0.05), and a similar potentiating effect was seen when 5-LOX activity was inhibited with 0.5 µmol/l REV 5901 (1.6 ± 0.2-fold increase above glucose plateau; means ± SE, n = 3, P < 0.05). In addition, inhibition of endogenous arachidonic acid metabolism by NS-398 or baicalein resulted in both a significant elevation in [3H]arachidonic acid release (53.4 ± 3% increase over efflux at 2 mmol/l glucose, P < 0.02, n = 3) and a rapid stimulation of insulin secretion at 2 mmol/l glucose (Fig. 3B). Furthermore, arachidonic acid–induced insulin release at basal (2 mmol/l) glucose levels was further enhanced by the addition of NS-398 and by phenidone, a dual COX and LOX inhibitor (Fig. 3C). Selective inhibition of COX-1 with 100 µmol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion from perifused human islets (1.33 ± 0.32-fold increase above glucose plateau; means ± SE, n = 3, P > 0.2).


Figure 3
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  		FIG. 3. Effect of exogenous arachidonic acid and COX and LOX inhibition on insulin secretion from human islets. A: Human islets were perifused with physiological salt solution containing 2 mmol/l glucose for the first 10 min and then with 20 mmol/l glucose alone for the next 20 min, followed by 20 mmol/l glucose in the presence of 50 µmol/l arachidonic acid ({diamondsuit}), 10 µmol/l NS-398 (•), or 1 µmol/l baicalein ({blacksquare}). Results are expressed as a percentage of basal insulin secretion at 2 mmol/l glucose (means ± SE, n = 3). B: Human islets were perifused with physiological salt solution containing 2 mmol/l glucose for the first 10 min and then with 2 mmol/l glucose supplemented with 50 µmol/l arachidonic acid ({diamondsuit}), 10 µmol/l NS-398 (•), or 1 µmol/l baicalein ({blacksquare}). Results are expressed as a percentage of basal insulin secretion at 2 mmol/l glucose (means ± SE, n = 3). C: Human islets were perifused with physiological salt solution containing 2 mmol/l glucose for the first 10 min and then with 2 mmol/l glucose supplemented with 50 µmol/l arachidonic acid (•, {square}) for the next 20 min, followed by 50 µmol/l arachidonic acid in the presence of 10 µmol/l NS-398 (•) or 50 µmol/l phenidone ({square}). Results are expressed as a percentage of basal insulin secretion at 2 mmol/l glucose (means ± SE, n = 3).

 
Effects of PGE2 on insulin secretion from human islets.
PGE2 (1 µmol/l) did not significantly inhibit insulin secretion from perifused human islets when administered directly with 20 mmol/l glucose (data not shown) or 50 µmol/l arachidonic acid (data not shown), nor did it inhibit insulin secretion when islets were pre-perifused with 1 µmol/l PGE2 before challenge with 20 mmol/l glucose (Fig. 4A). The lack of inhibitory effect of PGE2 on insulin secretion from human islets did not result from a lack of expression of appropriate receptors for this prostaglandin because mRNAs for all members of the PGE2 receptor family (EP1, -2, -3, and -4) could be detected in human islet cDNA (Fig. 4B). In addition, exposure of islets to PGE2 at 2 mmol/l glucose resulted in a significant stimulation of insulin release (Fig. 4A, cumulative insulin secretion in the presence of PGE2, 15.3 ± 2.8 vs. 7.1 ± 1.0 ng insulin/10 min in the absence of PGE2, n = 4, P < 0.05), indicating that EP receptors are functionally coupled in human islets. The data shown in Fig. 4A suggest that PGE2 may enhance glucose-stimulated insulin secretion, but this can be entirely accounted for by the higher rate of basal secretion induced by 1 µmol/l PGE2 at 2 mmol/l glucose. Static incubations of human islets also demonstrated that 1 µmol/l PGE2 did not affect glucose-stimulated insulin secretion (108 ± 6.7% of 20 mmol/l glucose-stimulated response, means ± SE, n = 8, P > 0.2). In the same experiments, PGE2 induced a significant inhibition of glucose-stimulated cyclic AMP generation (47 ± 3.8% of 20 mmol/l glucose-stimulated response, means± SE, n = 8, P < 0.05).


Figure 4
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  		FIG. 4. Effect of PGE2 on insulin on secretion from human islets. A: Human islets were perifused with physiological salt solution containing 2 mmol/l glucose for 20 min then with 20 mmol/l glucose for 20 min ({circ}). In the same experiments, islets were perifused with 2 mmol/l glucose for 10 min, with 1 µmol/l PGE2 at 2 mmol/l glucose for a further 10 min, and with 1 µmol/l PGE2 at 20 mmol/l glucose for the final 20 min (•). Results are expressed as nanograms insulin secreted per milliliter (means ± SE, n = 4). Insulin secretion was not significantly different at 2 mmol/l glucose before perifusion with 1 µmol/l PGE2 (•, 1.42 ± 0.27 ng insulin/ml; {circ}, 1.22 ± 0.23, P > 0.2). B: mRNAs coding for the PGE2 receptor subtypes EP1, EP2, EP3, and EP4 were detected in human islets by RT-PCR.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is no doubt that arachidonic acid and its metabolites play important roles in the regulation of ß-cell function, but most studies to date have used rodent islets or insulin-secreting cell lines, and little information is available about their functions in human ß-cells. As an essential prerequisite to investigating the role(s) of arachidonic acid in human islets, we investigated the expression profile of enzymes involved in arachidonic acid generation and metabolism. RT-PCR amplifications clearly indicated that mRNAs for cPLA2 and COX-1 and COX-2, but not COX-3, were detected in human islets. We and others have previously reported that human islets express COX-1 and COX-2 mRNAs and that COX-2 expression is regulated in a glucose-sensitive manner (30,31). The expression of COX-2 by human islet ß-cells, detected in a recent study by immunohistochemistry (31), was confirmed here by single-cell RT-PCR, and this is the first report, to our knowledge, that human islet {alpha} cells do not express COX-2. The presence of COX-2 in ß-cells and its upregulation in a glucose-dependent manner provide circumstantial evidence for this enzyme playing an important role in ß-cell function. Most of the data on islet LOX expression have been obtained in studies using rodent islets or cell lines, and there is convincing evidence that 12-LOX is the major LOX enzyme in rodent ß-cells (15,20,32). There has been a report of 5-LOX mRNA expression in rat islets (33), but the role of this isoform in rodents is thought to be negligible because the product of 5-LOX activation, 5-HETE, could not be detected in rat islets (20). A recent report has indicated that human islets express 12-LOX (16), and our RT-PCR analyses confirm this and indicate that they also express mRNA for 5-LOX, but not 15-LOX, suggesting close concordance between rat and human islets in LOX expression.

Our measurements of the time course of insulin secretion from perifused human islets support an important role for arachidonic acid in secretory responses to glucose. Thus, application of PLA2 inhibitors to inhibit endogenous arachidonic acid generation significantly decreased the amplitude of the insulin secretory response to 20 mmol/l glucose. A similar inhibitory effect of AACOCF3 on glucose-stimulated insulin secretion from rat islets has been reported previously (34), and these data are consistent with a stimulatory role for arachidonic acid or an arachidonic acid metabolite(s) in nutrient-regulated insulin secretion. The trifluoro methyl ketone arachidonic acid derivatives used in this study also stimulated basal insulin secretion from human islets. Similar stimulatory effects of AACOCF3 on insulin secretion from rat islets (33) and cytosolic calcium in HIT insulinoma cells (35) have been reported, and this may reflect the similarity in structure of PACOCF3 and AACOCF3 to arachidonic acid, supportive of a direct effect of arachidonic acid (rather than its metabolites) in the regulation of insulin secretion.

We have previously reported that arachidonic acid stimulates insulin release from isolated human islets (36). In the current study, we have examined whether elevation of endogenous arachidonic acid by inhibition of its metabolism through particular COX and LOX pathways led to stimulation of insulin secretion and whether the capacity of exogenous arachidonic acid to stimulate insulin secretion was affected by inhibition of its metabolism. Our data confirm that arachidonic acid stimulates insulin secretion from human islets, and we further demonstrate that inhibition of arachidonic acid metabolism through 5-LOX (0.5 µmol/l REV 5901), 12-LOX (1 µmol/l baicalein), and COX-2 (10 µmol/l NS-398) produces similar secretory profiles to those seen after addition of exogenous arachidonic acid, which suggests that the primary effects of COX and LOX inhibition may be to maintain elevated arachidonic acid levels. This was confirmed by perifusion experiments with [3H]arachidonic acid–prelabeled islets, which showed increases in [3H]arachidonic acid release in response to COX and LOX inhibition. Insulin secretion was not modified by acetaminophen when it was used at a concentration (100 µmol/l) to selectively inhibit COX-1 (10), suggesting that this enzyme is not a major source of arachidonic acid metabolism in human ß-cells. The predominance of COX-2–mediated signaling in the regulation of insulin secretion over that of COX-1 may reflect that the islets were maintained in culture medium containing 25 mmol/l glucose for up to 48 h before the secretion experiments: We have previously reported that COX-2 mRNA levels are upregulated approximately sevenfold when human islets are maintained at 25 mmol/l glucose (30). In addition to potentiating insulin secretion from human islets, we found that arachidonic acid, NS-398, and baicalein also initiated insulin secretion at 2 mmol/l glucose. Furthermore, exposure of human islets to NS-398 or phenidone, a global COX and LOX inhibitor, in the presence of arachidonic acid resulted in a significant stimulation of insulin secretion after the islets had already shown an initial secretory response to exogenous arachidonic acid. Taken together, these data indicate that blockade of arachidonic acid metabolism through COX-2 and LOX pathways stimulates basal, nutrient-, and arachidonic acid–induced insulin secretion from human islets and suggest that it is arachidonic acid rather than its metabolites that play a stimulatory role in the insulin secretory response.

Inhibition of COX-2 metabolism of arachidonic acid will reduce islet prostaglandin production, so we assessed whether this could account for the elevated insulin secretory output in the presence of NS-398 by measuring the effects of exogenous prostaglandin on insulin secretion. The exact metabolites synthesized by COX enzymes appear to be dependent on the cell type and stimulus: In the case of islets, PGE2 is reported to be the major prostaglandin synthesized. PGE2 did not significantly inhibit glucose-stimulated insulin secretion from human islets in static incubations or in perifusions. The presence of PGE2 receptor mRNAs, the stimulation of basal insulin secretion by PGE2, and the inhibition of cyclic AMP generation all indicated that human islets possess the appropriate machinery for sensing and responding to PGE2. Our observation of a stimulation of basal insulin secretion by PGE2 is consistent with a previous report in which PGE2 was shown to stimulate insulin secretion from human islets in static incubations (13) and suggests a fundamental difference in PGE2 action between rodent and human ß-cells. In rodent islets, PGE2 is thought to inhibit glucose-stimulated insulin secretion through activation of EP3 receptors that are linked to inhibition of adenylate cyclase (37). Our data indicate that human islets are also equipped with EP3 receptors through which PGE2 can decrease cyclic AMP generation. However, the expression by human islets of other PGE2 receptor family members that are associated with stimulation of second messenger generation may explain why PGE2 did not inhibit glucose-induced insulin secretion and stimulated basal insulin secretion from human ß-cells. With regard to this, PGE2 may be operating through EP1 receptors to elevate intracellular Ca2+, as has been observed in other cell types (38), and agonists that increase Ca2+ in human islets can initiate insulin secretion at substimulatory glucose concentrations (26,39,40).

Although it is clear that studies using rodent islets and insulin-secreting cell lines can provide information that is also appropriate to human islets (such as expression of COX and LOX isoforms), it is worth noting that there are some critical differences in the cPLA2/arachidonic acid/COX/LOX cascades between human and rodent islets. This is evident through observations that cPLA2 is expressed at higher levels in human islets than it is in rat islets (1), that PGE2 inhibits glucose-stimulated insulin secretion from rat islets and rodent insulin-secreting cell lines (12,14,21) but not from human islets (13) (data reported here), and that NS-398 inhibits the secretory response to glucose in rat islets (41), has no effect in HIT-T15 or ßHC13 cells (12), but enhances glucose-stimulated insulin release from human islets (data reported here). Thus, it would seem prudent to reproduce key observations in rodents using primary human islets wherever possible.

In summary, human islets express COX (COX-1 and COX-2) and LOX (5-LOX and 12-LOX) isoforms that have previously been detected in rodent islets. However, in contrast to data obtained with rat islets, inhibition of both LOX and COX pathways in human islets results in increased insulin secretion, consistent with arachidonic acid itself rather than one (or more) of its metabolites being responsible for the enhanced secretory response. Although arachidonic acid metabolism through COX and LOX pathways is not required for its stimulatory effects on insulin secretion from human islets, there is no doubt that COX-2 (12,13,37) and 12-LOX (15,16) play critical roles in cytokine-induced human ß-cell destruction, and selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: They would minimize ß-cell dysfunction while maintaining insulin secretory output through enhancing endogenous arachidonic acid levels.


   ACKNOWLEDGMENTS
 
D.M. has received grant support from The Eli Lilly International Foundation. V.D.B. has received grant support from The Eli Lilly International Foundation. I.K.M. has received a Medical Research Council PhD studentship. A.P. has received a King’s College London PhD studentship. C.J.B. was a Diabetes Research & Wellness Foundation research fellow.

We are grateful to H. Mandefield, the transplantation coordinators of South-Thames and King’s College Hospital, and relatives of the organ donors for human pancreata.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication April 12, 2006 and accepted in revised form September 19, 2006


   REFERENCES
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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