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The Role of Cytosolic Phospholipase A₂ in Insulin Secretion

From the Centre for Reproduction, Endocrinology and Diabetes, GKT School of Biomedical Sciences, King’s College London, London, U.K

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Cytosolic phospholipase A₂ (cPLA₂) comprises a widely expressed family of enzymes, some members of which have the properties required of signal transduction elements in electrically excitable cells. Thus, - and ß-isoforms of cPLA₂ are activated by the increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) achieved in depolarized cells. Activation is associated with a redistribution of the enzyme within the cell; activation of cPLA₂ generates arachidonic acid (AA), a biologically active unsaturated fatty acid that can be further metabolized to generate a plethora of biologically active molecules. Studies using relatively nonselective pharmacological inhibitors have implicated cPLA₂ in insulin secretory responses to stimuli that elevate ß-cell [Ca²⁺]_i; therefore, we have investigated the role of cPLA₂ in ß-cell function by generating ß-cell lines that under- or overexpress the -isoform of cPLA₂. The functional phenotype of the modified cells was assessed by observation of cellular ultrastructure, by measuring insulin gene expression and insulin protein content, and by measuring the effects of insulin secretagogues on cPLA₂ distribution, on changes in [Ca²⁺]_i, and on the rate and pattern of insulin secretion. Our results suggest that cPLA₂ is not required for the initiation of insulin secretion from ß-cells, but that it plays an important role in the maintenance of ß-cell insulin stores. Our data also demonstrate that excessive production of, or exposure to, AA is deleterious to normal ß-cell secretory function through metabolic dysfunction.

The ATP-sensitive group VIA PLA₂ enzyme, now designated iPLA₂ß, is an important islet PLA₂ species that is expressed in ß-cells (10,11). The iPLA₂ß enzyme does not require Ca²⁺ for catalytic activity and is inhibited by a bromoenol lactone (BEL) suicide substrate (11–14). It is activated by millimolar concentrations of ATP (in the presence of Mg²⁺) and inhibited by ADP (15); therefore, it is possible that the increased ATP/ADP ratio after nutrient metabolism may activate this enzyme, much in the same way as the ATP-sensitive K⁺ channels are thought to be sensitive to the ATP/ADP ratio rather than to ATP levels per se. The majority of studies on the role of iPLA₂ß in ß-cell function have been performed using rat islets and the HIT-T15 ß-cell line, but iPLA₂ß activity has also been quantified in human islets (10). An absolute requirement for iPLA₂ß in the physiological regulation of insulin secretion has not been established, but there are several lines of evidence to suggest that it plays an important role. Thus, the secretory response to glucose in rat islets and ß-cell lines is inhibited by BEL (11,16). Loss of glucose responsiveness of ß-cell lines is associated with a decline in iPLA₂ß activity (16), and potentiation of insulin release by cholecystokinin may be mediated, at least in part, through the activation of iPLA₂ß (17).

The type IV cytosolic phospholipase A₂ (cPLA₂) enzymes are also phospholipase activities that can be regulated by intracellular signals known to be important in regulating ß-cell function (18,19). Thus, cPLA₂ is a family of enzymes comprised of proteins of the predicted molecular masses of 85 kDa (cPLA₂), 114 kDa (cPLA₂ß), and 61 kDa (cPLA₂), all of which selectively hydrolyze membrane phospholipids to generate AA (20). Most importantly, the enzyme activity of the - and ß-isoforms of cPLA₂ is sensitive to micromolar concentrations of intracellular Ca²⁺ ([Ca²⁺]_i), suggesting that they may act as downstream effectors for the well-defined initial stages in ß-cell stimulus-secretion coupling, which result in increases in [Ca²⁺]_i. Furthermore, the discovery that cPLA₂ activity can also be regulated through phosphorylation (18,19) by protein serine/threonine kinases that have been implicated in stimulus-response coupling in ß-cells (21) offered multiple levels for the control of ß-cell cPLA₂ activity by insulin secretagogues. Given the importance of both [Ca²⁺]_i and protein phosphorylation in stimulus-response coupling in ß-cells, this article will focus on the possible role of cPLA₂ in the regulation of insulin secretion.

Most studies of cPLA₂ have focused on the physiological roles of the -isoform, which was the first member of the cPLA₂ family to be identified and which has been implicated in a range of cellular processes, including mitogenesis, allergic responses, fertility, and cytotoxicity (18,19). There is considerable circumstantial evidence that cPLA₂ plays an important role in regulating insulin secretion. Thus, cPLA₂ expression has been identified in islets and ß-cells by Western blotting, Northern blotting, and PCR (5,7,8), and ß-cell cPLA₂ enzyme activity has been shown to be sensitive to Ca²⁺ in the micromolar range (22). Glucose and other stimuli that increase ß-cell [Ca²⁺]_i activate Ca²⁺-dependent cytosolic PLA₂ in islets (22), and pharmacological inhibitors of cPLA₂ inhibit glucose-induced insulin secretion (5).

Studies using pharmacological inhibitors of PLA₂ cannot be considered as conclusive because of the overlapping specificities of the inhibitors for the many different classes of PLA₂ enzymes expressed in most cells. For example, earlier studies on insulin secretion using the then available PLA₂ inhibitors are difficult to interpret because many of these compounds have multiple effects, not always related to PLA₂ activity (1). Similarly, the newer classes of AA-like PLA₂ inhibitors do not differentiate between cPLA₂ and the Ca²⁺-independent PLA₂ activity, nor do they discriminate between the cPLA₂ isoforms (19). Furthermore, the BEL inhibitor of iPLA₂ß suppresses the effect of glucose on the ATP:ADP ratio in mouse islets (23), which may account for some of its inhibitory effects on glucose-induced insulin secretion (11,16). To circumvent the use of pharmacological inhibitors, we chose to investigate the role of cPLA₂ in ß-cells by generating ß-cell lines in which cPLA₂ was permanently underexpressed (23) or overexpressed by stable transfection with vectors encoding cPLA₂ in the antisense or sense orientation, respectively. Our studies using these modified ß-cells suggest that the activation of cPLA₂ is not required for glucose-induced insulin secretion, in contrast to the available circumstantial evidence.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Human islets of Langerhans were obtained with appropriate consent from the Human Islet Transplantation Unit at King’s College London (via Dr. G.-C. Huang and Prof. S. Amiel). Rat islets were isolated by collagenase digestion of whole rat pancreas. MIN6 cells were obtained from Dr. Y. Oka and Prof. J.-I. Miyazaki (University of Tokyo) and stably transfected with vectors encoding the full-length mouse cPLA₂ cDNA (a gift from Dr. B. Kennedy, Merck Frosst, PQ, Canada) in the pcDNA3.1 vector (Stratagene Europe, Amsterdam, the Netherlands) as described (24). cPLA₂ immunoreactivity was detected by Western blotting (24) or fluorescence immunocytochemistry (25) using an anti-cPLA₂ antibody (Genetics Institute, Cambridge, MA). PLA₂ enzyme activity was measured by quantifying the release of [³H]-AA from the sn-2 position of phosphatidylcholine L-stearoyl-2-[³H]-arachidonyl (³H-PC). Briefly, ³H-PC (2 µmol/l, 4 Ci/mmol) was incubated (10 min, 37°C) with tissue extracts in a final volume of 25 µl Tris buffer (pH 8.0) containing 50% glycerol (vol/vol), 1 mmol/l dithiothreitol, 0.5 mmol/l NaVO₄, 0.25 mmol/l NaF, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 20 µmol/l E64, 20 µmol/l tosyl-L-lysine chloromethyl ketone, 10 µmol/l okadaic acid, 0.1% ß-mercaptoethanol (vol/vol), and 50 µg/ml leupeptin and supplemented with either 0.8 mmol/l CaCl₂ or 1 mmol/l EGTA. Reactions were terminated, and products were extracted by the addition of butanol (40 µl, 4°C). [³H]-AA was separated from an uncleaved substrate by thin-layer chromatography on silica thin-layer gels using petroleum ether:diethyl ether:glacial acetic acid 70:30:1 (vol/vol/vol) and quantified by liquid scintillation counting. Changes in [Ca²⁺]_i in MIN6 cells were assessed by single-cell microfluorimetry of Fura-2 loaded cells (26). Changes in intracellular NAD(P)H were assessed by measuring NAD(P)H autofluorescence in MIN6 cell suspensions. The rate and pattern of insulin release was assessed using a multi-chamber perifusion system at 37°C in a temperature-controlled environment (24). To maximize secretory responses, MIN6 cells were configured as pseudoislets for secretion experiments (27). Insulin content was measured by radioimmunoassay (28), and quantitative measurements of (pre)proinsulin mRNA were obtained using real-time quantitative PCR with GAPDH as an internal standard (29). Electron micrographs were prepared by the Electron Microscope Unit at King’s College London using standard techniques. Data are expressed as means ± SE and were analyzed using ANOVA, Student’s t test, and Bonferroni’s multiple comparison test as appropriate. Differences between treatments were considered significant at P < 0.05.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Expression, distribution, and translocation of cPLA₂ in ß-cells.
As has been reported previously (5,7,8), islets and ß-cell lines express cPLA₂ immunoreactivity and enzyme activity. Figure 1A shows immunoreactive cPLA₂ in extracts of the mouse MIN6 insulin-secreting cell line (lanes 1 and 3) and human islets (lane 2). Whereas MIN6 cells contain only one immunoreactive protein migrating as cPLA₂, human islets also express another lower molecular weight immunoreactivity that may represent the recently identified cPLA₂ isoform (20). At present, it is not known whether this lower molecular weight immunoreactivity is expressed in human ß-cells or in another islet cell type. The cPLA₂ enzymes are so named because, in many cell types, they are primarily localized in the cytosol, and enzyme activity is regulated through Ca²⁺-dependent translocation to membrane compartments enriched in the phosphatidylcholine substrate (18,30). Figure 1B shows Ca²⁺-dependent and Ca²⁺-independent PLA₂ enzyme activities in fractions prepared from unstimulated rat islets, where the EGTA-inhibited activity most likely reflects the Ca²⁺-dependent type IV cPLA₂ activity. The Ca²⁺-insensitive PLA₂ activity, detected in the presence of EGTA, was largely confined to the cytosol, whereas the Ca²⁺-sensitive cPLA₂ activity was found in both the cytosolic and particulate fractions.

View larger version (40K): [in this window] [in a new window]   FIG. 1. cPLA2 expression, activity, and distribution in ß-cells. A: Western blotting with an anti-cPLA2 antiserum confirmed the expression of the protein in a mouse insulin-secreting cell line (MIN6 cells) and in an extract of whole human primary islets. B: The generation of AA from phosphatidylcholine was measured to estimate the Ca2+-dependent and Ca2+-independent PLA2 activity in fractions prepared from whole rat islets. Type I and II PLA2 activities were inhibited by the presence of DDT and ß-mercaptoethanol in the assay. , Total activity (Ca2+-dependent and Ca2+-independent) in the presence of Ca2+. , Ca2+-independent activity measured in the absence of Ca2+ and the presence of EGTA (1 mmol/l). The Ca2+-dependent type activity is the difference between these values. Bars show means ± SE (n = 4). C: Visualizing cPLA2 distribution by fluorescence immunocytochemistry with an anti-cPLA2 antiserum demonstrated that the enzyme was distributed throughout the cytosolic and nuclear compartments in unstimulated MIN6 cells (left panel, 2 mmol/l glucose) and translocated from the nucleus in response to the increases in [Ca2+]i caused by exposure to tolbutamide (100 µmol/l, 30 min). D: AA (50 µmol/l, arrow) stimulated insulin secretion from human islets of Langerhans being perifused (0.5 ml/min, 37°C) with a physiological salt solution supplemented with 0.5 mg/ml BSA and 2 mmol/l glucose. Points show means ± SE for four separate perifusion channels.

Although unusual, this distribution of cPLA₂ is not unique to ß-cells because bovine endothelial cells are reported to exhibit a basal nuclear localization of cPLA₂, which translocates from the nucleus in response to Ca²⁺-mobilizing stimuli (31). We suggest that the redistribution of cPLA₂ in stimulated ß-cells may allow agents that increase [Ca²⁺]_i to rapidly increase cPLA₂ activity in the cytoplasm by promoting its translocation from the nucleus, in a similar manner to the regulation of hepatic glucokinase by its export from the nucleus to the cytoplasm (32,33). Whatever the functional significance of the Ca²⁺-induced translocation of ß-cell cPLA₂ from the nucleus, it is consistent with a stimulus-dependent activation of the enzyme, and the subsequent generation of AA, being involved in stimulus-secretion coupling. In support of this chain of events, Fig. 1D shows that exogenously applied AA (50 µmol/l) stimulated insulin secretion from human islets in the presence of a substimulatory concentration of glucose (2 mmol/l), suggesting that elevated levels of AA are sufficient to initiate a secretory response.

Functional phenotype of cPLA₂-deficient ß-cells.
MIN6 cells deficient in cPLA₂ were generated by transfection with a vector coding cPLA₂ in the antisense orientation and selection for G418-resistant clones. A number of stably transfected clones showing a loss of cPLA₂ expression by immunoblotting were expanded and used for functional studies, showing a stable cPLA₂-deficient phenotype for a least 1 year in continuous culture (24). The cPLA₂-deficient cells had a much lower basal (2 mmol/l glucose) rate of insulin secretion per cell (8.6 ± 2% of passage-matched controls, P < 0.01, n = 4). However, the ability of the cells to recognize and respond to elevations in the extracellular glucose concentration was not markedly affected by the loss of cPLA₂, as shown in Fig. 2A. In these perifusion experiments, the secretory responses of cPLA₂-deficient cells to increased glucose were similar to those of control cells when expressed relative to the basal secretory rate, showing a rapid increase in insulin release upon elevation of glucose from 2 to 20 mmol/l and maintaining an elevated rate of secretion thereafter. The cPLA₂-deficient cells also showed normal patterns of insulin secretion in response to nonnutrient stimuli that elevate [Ca²⁺]_i, such as tolbutamide and KCl (24). These data imply that cPLA₂ is not required for pancreatic ß-cells to show an initial secretory response to nutrient and nonnutrient secretagogues, suggesting that it is not a pivotal Ca²⁺-sensitive sensor in ß-cell stimulus-secretion coupling. It has been reported previously that the amplifying (ATP-dependent K⁺ channel-independent) effect of glucose on insulin secretion from mouse islets does not require PLA₂ activation and AA generation (23); therefore, it seems unlikely that cPLA₂ plays an important role in glucose-induced insulin secretion.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Functional phenotype of cPLA2-deficient ß-cells. A: MIN6 pseudoislets formed from cPLA2-deficient () or passage-matched control () cells were perifused (0.5 ml/min, 37°C) with a physiological salt solution supplemented with 0.5 mg/ml BSA and 2 mmol/l glucose to establish a stable basal rate of secretion. Exposure to 20 mmol/l glucose (arrow) produced a rapid and maintained elevation in insulin secretion from both populations. Points show means ± SE (n = 3). B: After the exposure to 20 mmol/l glucose in A, the pseudoislets were exposed to the adenylate cyclase activator forskolin (10 µmol/l in the presence of 100 µmol/l isobutylmethylxanthine), as shown, in the continued presence of 20 mmol/l glucose. , Means ± SE, n = 3; where error bars are not shown, SEs were smaller than the symbols. , The means of two experiments.

It is not immediately apparent why cPLA₂ deficiency should cause such marked reductions in insulin content, but it cannot be accounted for by reduced insulin gene transcription because quantitative RT-PCR demonstrated similar levels of preproinsulin mRNA in cPLA₂-deficient and control cells (cPLA₂-deficient 84 ± 16% control, n = 3), suggesting a defect in translation, processing, or packaging of (pre)proinsulin. In other tissues, cPLA₂ has been implicated in maintaining the stability of the Golgi apparatus to enable appropriate intracellular vesicle trafficking (34), and this would be consistent with reported changes in ultrastructure of cPLA₂-deficient MIN6 cells (24). Thus, in accordance with their reduced insulin content, these cells contained very few typical dense-cored insulin-containing secretory vesicles and large numbers of nonelectron dense vesicles (24). Further studies are required to determine whether the reduced content of insulin-containing dense-cored secretory vesicles is due to defective packaging of the vesicles or to defective processing and crystallization of insulin.

In summary, the functional phenotype of ß-cells deficient in cPLA₂ does not support a vital role for cPLA₂ as a Ca²⁺-sensitive transduction element in the ß-cell stimulus-secretion coupling pathway, but implicates cPLA₂ in the maintenance of insulin stores in dense-cored secretory vesicles. These conclusions are not entirely inconsistent with some of the earlier studies that provided the rationale for the current work. Thus, physiological stimuli that enhance insulin secretion must also enhance the production and storage of more insulin to maintain the ß-cell secretory capacity. The activation/redistribution of cPLA₂ in response to insulin secretagogues may be involved in this element of stimulus-response coupling, rather than in the immediate regulation of the secretory process.

Functional phenotype of cPLA₂-overexpressing ß-cells.
To complement our studies on cPLA₂-depleted ß-cells, we have started to investigate the functional consequences of constitutive overexpression of cPLA₂ by generating stably transfected MIN6 cells expressing the full-length mouse cDNA inserted into the pcDNA3.1 expression vector in the sense orientation. Overexpression of cPLA₂ was driven by the cytomegalovirus promoter and produced high and stable levels of expression of immunoreactive cPLA₂, as assessed by Western blotting. As for the cPLA₂-deficient MIN6 cells, the functional phenotype of the overexpressing clones was not what we had expected. Thus, overexpression of cPLA₂ had no marked effect on insulin content nor on the basal rate of insulin secretion at a substimulatory glucose concentration (2 mmol/l). However, the cPLA₂-overexpressing clones failed to show insulin secretory responses to glucose, as shown in Fig. 3A. The lack of secretory responses to glucose was accompanied by a loss of the normal rapid increases in [Ca²⁺]_i induced by glucose (data not shown) and by mitochondrial substrates such as ketoisocaproic acid, as shown in Fig. 3B, perhaps suggesting a mitochondrial defect. The cells retained normal [Ca²⁺]_i responses to depolarizing concentrations of KCl (Fig. 3B) and tolbutamide (data not shown), localizing the defect in the stimulus-response coupling pathway at a stage beyond glucose entry and phosphorylation but before depolarization of the ß-cells and the subsequent influx of extracellular Ca²⁺. This conclusion was supported by the demonstration that cPLA₂-overexpressing cells had relatively normal, although slightly delayed, onset and secretory responses to depolarizing [Ca²⁺]_i-elevating stimuli, as shown in Fig. 3C. Incidentally, the normal pattern of both basal secretion and of secretion in response to elevated [Ca²⁺]_i in cells that greatly overexpress cPLA₂ further reinforces the conclusion from the studies with cPLA₂-deficient MIN6 cells that cPLA₂ is not an important transducer of Ca²⁺-induced secretory responses in ß-cells.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Functional phenotype of cPLA2-overexpressing ß-cells. A: MIN6 pseudoislets formed from cPLA2-overexpressing () or passage-matched control () cells were perifused (0.5 ml/min, 37°C) with a physiological salt solution supplemented with 0.5 mg/ml BSA and 2 mmol/l glucose to establish a stable basal rate of secretion. Exposure to 20 mmol/l glucose (arrow) produced a rapid and maintained elevation in insulin secretion from control pseudoislets as shown in this representative experiment (), but the cPLA2-overexpressing cells failed to respond (, means ± SE, n = 3). B: Microfluorimetric measurements of [Ca2+]i in Fura2-loaded cPLA2-overexpressing MIN6 cells showed a loss of the normal rapid increases in [Ca2+]i induced by nutrients such as ketoisocaproic acid (KIC, 10 mmol/l), although the cells showed normal [Ca2+]i responses to tolbutamide (Tolb, 100 µmol/l) and to KCl (20 mmol/l). C: Representative experiments in which a depolarizing concentration of KCl (20 mmol/l arrow) stimulated insulin secretion from pseudoislets formed from cPLA2-overexpressing () or passage-matched control () MIN6 cells.

In conclusion, our expectation at the outset of these studies was to confirm an important role for cPLA₂ as a Ca²⁺ sensor in ß-cell stimulus-secretion coupling. However, our results did not support our preconceptions and suggest that cPLA₂ is not required for Ca²⁺-dependent insulin secretion, although it may play an important, but poorly defined, role in the maintenance of insulin stores in dense-cored secretory vesicles. The unexpected phenotype associated with excessive cPLA₂ activity may give further insights into fatty acids, mitochondrial dysfunction, and the failure of ß-cell secretory responses.

	ACKNOWLEDGMENTS

 
We are grateful to Diabetes UK for project grant support (RD97/0001525). C.J.B. is a Diabetes Research and Wellness Foundation fellow. V.D.B. was funded by the Eli Lilly International Foundation. H.M.R.-M. was funded by a Biotechnology and Biological Sciences Research Counsel Animal Sciences Committee postgraduate studentship.

We are grateful to Dr. Paul Squires (Warwick University, U.K.) for measurements of [Ca²⁺]_i, to Dr. K.J. Parker (King’s College London) for the PLA₂ enzyme assay, and to Nicholas Evans (King’s College London) for measurements of NAD(P)H autofluorescence in MIN6 cells. We thank Dr. Y. Oka and Prof. J.I. Miyazaki (University of Tokyo) for provision of the MIN6 cells, Dr. B. Kennedy (Merck Frosst, PQ, Canada) for the cPLA₂ cDNA, Dr. R. Kramer (Eli Lilly, Indianapolis, IL) for the cPLA₂ antiserum, and Dr. G.-C. Huang and Prof. S. Amiel (King’s College London) for the human islets.

	FOOTNOTES

 
This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible by an unrestricted educational grant from Les Laboratoires Servier.

Address correspondence and reprint requests to P.M. Jones, Room 3.2A, New Hunt’s House, CRED, GKT School of Biomedical Sciences, King’s College London, London SE1 1UL, U.K. E-mail: peter.jones{at}kcl.ac.uk

Received for publication March 13, 2003 and accepted in revised form May 1, 2003

Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone; [Ca²⁺]_i, intracellular Ca²⁺ concentration; cPLA₂, cytosolic phospholipase A₂; PLA₂, phospholipase A₂; UPC2, uncoupling protein 2

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