Cyclooxygenase-2, not microsomal prostaglandin E synthase-1, is the mechanism for interleukin-1β-induced prostaglandin E2 production and inhibition of insulin secretion in pancreatic islets

Abstract

Arachidonic acid is converted to prostaglandin E(2) (PGE(2)) by a sequential enzymatic reaction performed by two isoenzyme groups, cyclooxygenases (COX-1 and COX-2) and terminal prostaglandin E synthases (cPGES, mPGES-1, and mPGES-2). mPGES-1 is widely considered to be the final enzyme regulating COX-2-dependent PGE(2) synthesis. These generalizations have been based in most part on experiments utilizing gene expression analyses of cell lines and tumor tissue. To assess the relevance of these generalizations to a native mammalian tissue, we used isolated human and rodent pancreatic islets to examine interleukin (IL)-1β-induced PGE(2) production, because PGE(2) has been shown to mediate IL-1β inhibition of islet function. Rat islets constitutively expressed mRNAs of COX-1, COX-2, cPGES, and mPGES-1. As expected, IL-1β increased mRNA levels for COX-2 and mPGES-1, but not for COX-1 or cPGES. Basal protein levels of COX-1, cPGES, and mPGES-2 were readily detected in whole cell extracts but were not regulated by IL-1β. IL-1β increased protein levels of COX-2, but unexpectedly mPGES-1 protein levels were low and unaffected. In microsomal extracts, mPGES-1 protein was barely detectable in rat islets but clearly present in human islets; however, in neither case did IL-1β increase mPGES-1 protein levels. To further assess the importance of mPGES-1 to IL-1β regulation of an islet physiologic response, glucose-stimulated insulin secretion was examined in isolated islets of WT and mPGES-1-deficient mice. IL-1β inhibited glucose-stimulated insulin secretion equally in both WT and mPGES-1(-/-) islets, indicating that COX-2, not mPGES-1, mediates IL-1β-induced PGE(2) production and subsequent inhibition of insulin secretion.

FIGURE 1.

Stimulation by IL-1β of PGE₂ production by rodent and human islets. Cultured human islets produced increasing levels of PGE₂ in the presence of IL-1β in a time-related, linear manner, and PGE₂ production stimulated by IL-1β was completely inhibited by the specific COX-2 antagonist, NS-398 (p < 0.01–0.001). Islets were obtained from three different donors; individual experiments were performed in triplicate. Inset, 24-h incubation results from experiments using mice and rat isolated islets were comparable with the experiments using human islets, i.e. NS-398 inhibited IL-1β-induced PGE₂ production (*, p < 0.05; **, p < 0.01).

FIGURE 2.

Basal and stimulated levels of COX-1, COX-2, cPGES, and mPGES-1 mRNAs in rat pancreatic islets incubated with or without IL-1β. mRNA levels were quantified by triplicate determinations using real-time RT-PCR, and data were normalized to levels of islet COX-1 mRNA. Levels of COX-1 mRNA were similar under basal and IL-1β-stimulated conditions, whereas COX-2 mRNA levels after stimulation with IL-1β were higher than their basal levels (n = 7 pairs, p < 0.01). cPGES mRNA levels under basal and IL-1β-stimulated conditions were similar, whereas mPGES-1 mRNA levels after stimulation with IL-1β were higher than basal levels (n = 7 pairs, p < 0.02).

FIGURE 3.

Western analysis using 25 μg of protein of whole cell extracts of rat, mouse, and human islets for basal and IL-1β-stimulated levels of COXs and PGESs. COX-1 protein levels did not increase with IL-1β, whereas COX-2 protein levels did. cPGES and mPGES-2 protein levels were present in the nonstimulated state, and neither was stimulated by IL-1β. mPGES-1 was not detectable with or without stimulation by IL-1β.

FIGURE 4.

Summary of results from Western analysis of islets using whole cell extracts (25 μg of protein). A and B, islets from three different rats are shown. A, COX-1 levels did not increase with IL-1β, whereas COX-2 levels did (p < 0.05). B, levels of cPGES and mPGES-2 proteins were readily detectable but did not increase with IL-1β stimulation. C and D, islets from three different mice are shown. C, COX-1 levels did not increase with IL-1β, whereas COX-2 levels did (p < 0.05). D, levels of cPGES and mPGES-2 proteins were detectable but did not increase with IL-1β stimulation. E and F, human islets from three different donors are shown. E, constitutive levels of COX-1 and COX-2 were detectable, but only COX-2 levels were higher after exposure to IL-1β (p < 0.057). F, cPGES levels were not prominent in either the nonstimulated or stimulated state. Levels of mPGES-2 protein were prominently detected in the nonstimulated state but did not increase with IL-1β stimulation (N.D., nondetectable).

FIGURE 5.

Western analyses for mPGES-1 using microsomal versus whole cell extracts. Top panel, rat islets are shown. Whole cell extracts and microsomal extracts using greater amounts of protein than were used in Fig. 3 failed to reveal stimulation of mPGES-1 levels by IL-1β. Bottom panel, human islets are shown. mPGES-1 was detectable in whole cell extracts using greater amounts of protein than were used in Fig. 3 and revealed mPGES-1, but these levels were not increased when islets were treated with IL-1β.

FIGURE 6.

Inhibitory effects of IL-1β on glucose-induced insulin secretion from isolated islets of wild type and mPGES-1-deficient mice. Insulin secretion from wild type islets incubated in buffer containing 2.8 mm glucose was unaffected by IL-1β. In contrast, when a stimulatory concentration of glucose (16.7 mm) was used, IL-1β significantly inhibited insulin secretion (p < 0.05–0.01). Identical findings were observed when islets from mPGES-1-deficient mice were used. Experiments with epinephrine (epi), somatostatin (som), and PGE₂ are also shown for both wild type and mPGES-1 KO mice as comparators for the efficacy of IL-1β as an inhibitor of insulin secretion.

FIGURE 7.

General scheme illustrating the arachidonic acid pathway through which IL-1β regulates PGE₂ production. Arachidonic acid is cleaved from phospholipid by phospholipase A₂ (PLA₂), followed by synthesis of endoperoxides (PGG₂, PGH₂) via COX-1 and COX-2. Activation of COX-2 by IL-1β increases intracellular PGE₂ levels. This scheme emphasizes that islets differ importantly from other cell lines and tumor tissues, because they contain little to no mPGES-1 in mice and rats, and although detectable in humans, this protein is not increased by IL-1β, whereas COX-2 is. Therefore, COX-2, and not mPGES-1, in islets is the mechanism whereby IL-1β increases PGE₂ production, which in turn inhibits glucose-induced insulin secretion.