Biocidmlca el Biol~Jydc.a.4cla. 11350992) 215-220
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g) 1992Elsev/er Scicn:e Pablishe~ g.v. All r/ghls rcserced 01674889/92/$05.00
BBAMCR 1317|
Inhibition of phospholipase A 2 and insulin secretion
in pancreatic islets R o b e r t J. K o n r a d , Y . C a m i l l e J o l l y , C h r i s t o p h e r M a j o r a n d B r y a n A . W o l f Dep~rtment of Palludogy and L~bomrory M~di¢itle. Unit ersily of Pennsyl~'ania Sdmol of Medicine. Philnde~hia, PA CUSA)
(R~cgived 19 September 1991) (Revisedmanuscriptr~ived 15 January 1992) Key ~ords: PhoxpholipaseA:: tnsciin ~crction; Isletsof l~ngerhans: Glucose;AracLidonicacid Arachidonic acid may be an imponant mediator of insulin s~cxetion since (D glucose activates phospbolipase A 2 thus increasing endogenous unesterified levels of arachidonic acid, (2) arachidonic acid mobilizes Caz+ from the islet endoplasmic retkulum and (3) ararhidoni¢ acid has been peolmsed to regulate voltage-dependent Ca2+ channels in the fl-¢,elL We have used ~he phospholip~ A 2 ioh~itur, (p-amytcinnamayl)anthranilic acid (ACA), to determine whether phespholipase A 2 activalion is requited for glucose*induced insulin secretion. ACA inhibited in a dose-dependent manner gioc~se-ioduced insulin secretion, as well as glyceraldehyde and a-ketoisocaproic acid-induced insulin secretion. ACA also tolally abolished glucase-ioduced arachidona~e accumulation but did not affect phospho[ipase C suggesting that it was specific for phospholipase A2. Furthermore, ACA did not inhibit g]u~se o~dation. These obselvatinas suggest tha~ glucose-induced arachido~ate increase is essential for insulin secretion.
Introduction D-GIncoSe is the main physiological stimulus of insulin st.~Cretinu by islets of Langerhans [1,7..]. The precise mechanisms L-,voE,ed are not completely elucidated but it is known that glucose metabolism is necessary for insulin secretion [3]. Glucose oxidation is believed to increase intraceilular A T P levels, ansi to close the KATe channels [4,5]. Closure of these channels ~epol~rlzes the ft--cell, which activates the voltage-dependent Ca 2÷ channels resulting in an inflt~ of extracellular C.,a2+, and insulin exocytnsis [6]. There has also been growing moognitiou that glucase activates fipid signal traasductinn pathways in the ~-ceil: in particular both phosphulipases C and A 2 are rapidly activated [7,8]. Glucose-induced activation of these phospholipesns has generally been considered to be mediated exclusively through an increase in intracellular Ca 2÷ [9]. Gluoose stimulation of arachidonic acid accumulation is mediated by phospholipase A 2
AbbreviaTioas: ACA. (p-a.~cmnamoyl~tanthranilic acid; DEDA. 7,7~din~thy1.5.8-clot,sadienoic acid. Carr~aondence: B.A- Wolf, Universityof Peansylvanla School of Medicine, Department of P a t h o ~ and Laboratory Medicine, 217 John Morgan,philadelphia,PA 19104-60~.,USA.
activation [10]. Wc have recently shown, however, that the glucose-induced increase in intracellular arachidonate has a Ca2+-independent component which, coupled with tl"e Ca2+-mobiliziog effects of araehidonatu on the islet endoplasmic retieuinm as well as its ability to regulate voltage-dependent Ca 2+ channels in the /l-ceil, suggest that arachidonic acid may be an important intracelinlar mediator of insulin secretion [11-14]. In order to implicate arachidonic acid as a second messenger in insulin secretion, it is necessary to demonstrate that, in addition to its documented intracellular effects, exogenous arachidonic acid sti:aulatns insulin secretion, and inhibition of gle,'~os¢ induced arachidonic acid accumulation correlates with inin'bitinn of insulin secretion. Demonstration of arachidonic-induced insulin secretion has been rather arduous due in part Io the large endogenous amount of free uncstcdfied arachidonic acid present under normal conditions as well as solubility problems. Recently, Melt has dearly shown that exogenous arachidonlc acid, in a concentration range found inside /3-ceils, promotes insulin secretion, and has suggested that arachidonic acid LS a complete secretagogu¢ [10,15]. Other investigators have also suggested that arachidonic acid may be a second messenger [16]. In the present study, we have examined the effects of two purported inhibitors of phaspholipase A2, ACA [17] and DEDA [18], on glucose.induced insulin secretion.
216 E~mnSemttaJ w ~ d e r e s
Materinis Male vims-fsee Spragne-Dawley rats (200-250 g) were purchased from Charles-River (Wilmington, MA). Collagenase P (Lots 20/12 and 23/24) was obtained from Boehringer-Mannheim Corporation (Indianapolis, IN). Tissue culture medium (CMRL-1066) and 1 M Hepes were from Gibco (Grand Island, NY). Newborn bovine serum was from Hazleton BiOogics (Lcoean, KS). The following c:~mpounds were purchased from Sigma (St. Louis, MO):. D-glucose, Hanks" balanced salt ~ u t i o n , ~sen~'llin, streptomycin, glutamine, Ficoll, bovine a~bomin (RIA grade, fraction V). ACA (Lot M3222) and D E D A were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Pbasplmlipid standards were obtained from Serdary Research L a b o r a t ~ (Port H mo n , MI). Nan~ral fipids and fatty a d d s were purchased from Nu Chek Prep (Elysian, MN). Organic solvents (HPLC grade or Op6 m a grade)were pt'ovided by Fisher (Pittsburgh, PA). Other chemicals (except as indicated) were porchased from Sigma or Wisher. [5,6,8,9,11,12,14,15-3H(N)] Arachidonic acid (180-240 C i / m m o l ) was ob~ined from American Radinlabeled Company (St. Lonis, MO). The liquid scintillation cocktail Universol was lmm:hased from ICN Biomedicals (Costa Mesa, CA). [uC(U)]Glucose (290 m C i / m m o l ) was obtained from New England Nuclear (Bor~on, MA).
Me~ods Is~at/on of/s/ets. In a typ;cal experiment, islets were isolated aseptically born 8-10 male Sprague-Dawiey rats [19,20]. In brief, the bile duct was cannulated and the pancreas was inflated with appreu~. 20 ml of Hanks" balanced selt solution supplemented with penicillin/.25 U / m l ) and streptomycin (25 p g / m l ) - The distended pancreas was theo excised, and cleaned under a stereumlcroscope to remove lymph nodes, fat, blood vessels and bile duct. The tissue was then chopped, rinsed extensively with Hanks" solution, allowed to settle, and then digested with collagenase P (3-6 m g / m l of chopped tissue) at 39~C for 0.5 rain. The digested tissue was rinsed with Hanks" solution and then purified by centrifugation on a discontinuous Ficoll gradient (dialyzed and lyophdized; four layers of 27%, 23%, 20.5% and 11% in Hanks" Hepes (25 raM) buffer). The majority of the islets rose to the 11-20.5% interface and were collected, washed and then cleaned in 'complete' CMRL-1066 culture medium (supplemented with 10% heat-inactivated newborn bovine serum, 2 mM L-ghltunline, 50 U / I n l of penicillin and 50 p.g/ml of streptomycin, and containing 5.5 mM o-glucnse). This procedure typically provided a pure preparation of 350-400 islets pet rat [|9] which were then used as desoabod below.
Static incul~timl of islets. 25 freshly isolated islets were placed in siliconized 10 × 75 m m hernsilicate tubes, preinco~ated 30 rain at 37°C in 1 ml of fresh Krel~-Hepef medium (25 mM Hepes (pH 7.4), 115 mM NaCI, 24 mM NaHCO3, 5 m M KCI, 2.5 mM CaCI z, l mM MgCI 2, 0.1% bovine serum albumin) supplemented with 3 mM glucose and gassed with 95% 0 2 / 5 % CO2, and then incubated anmher 30 ndn with fresh medium containing 3 or 28 mM glu,:ose and the inhibitor (dissolved in a final concentration of 2% ethanol which was also present under the control conditions). Insulin cot_Lent in the supernetant was assayed by RIA [12,21L Peri~ of islets. Freshly isolated islets (200 per condition) were placed onto a mixed cellulose acetate and nitrate filter (Type SM, 5 p.m pore size, Milllpore Corporation, Bedford, MA) in a Swinnex 13 mm perifusion chamber (Milfipore) and allowed to eqnih'brate at a flow rate of I m l / m i n for 30 rain at 37°C in Krebs-Hepes medium supplemented with 3 mM glucose which was continually gassed with 95% 0 2 / 5 % C O 2. The medium was then changed to the experimental condition and perifusion was pursued for antuher 30 rain at a flow rate of I m l / m i n . The perifusate was collected every 2 rain in sificonized 12 3