Lysophosphatidylinositol, but not lysophosphatidic acid, stimulates insulin release

Lysophosphatidylinositol, but not lysophosphatidic acid, stimulates insulin release


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Vol. 138, No. 2, ! 9 8 6


Pages 720-727

July 31, 1986

LYSOPHOSPHATIDYLINOSITOL, B U T NOT LYSOPHOSPHATIDIC ACID, STIMULATES INSULIN RELEASE A Possible Rote for Phospholipase A 2 but Not de novo Synthesis of Lysophospholipid in Pancreatic Islet Function STEWART A. METZ University of Colorado Health Sciences Center (C-237) 4200 E. 9th Avenue Denver, Colorado 80262 and Denver VAMedica! Center, Denver, Colorado 80220 Received May 30, 1986

Summary: In the current study, lysophosphatidylinositol is shown to promote insulin release in a manner having characteristics of physiologic exocytosis--that is, it is dosedependent, saturable, reversible, inhibitable and unassociated with detrimental effects on subsequent islet functioning. Lysophosphatidylglycerol had similar insulinotropic effects. However, lysophosphatidic acid was ineffective over a wide range of concentrations. These studies provide further support for the postulated vole of phosphotipase Agenerated lysophospholipids in signal [ransduction in the pancreatic islet but suggest that any de novo synthesis of lysophosphatidic acid from metabotites of glucose (M. Dunlop and R. Larkins, F3iochem. BiophEs. Res. Comm. 132:467, 1985) is unlikely to contribute directly to the insulin secretion induced by that fuel. ® 1986AcademicPress, Inc. Recent interest in the possible vote of phospholipase A 2 (PLA 2) activation in the mechanism of glucose-induced insulin (I) release has generally focused oil one membrane hydrolysis product, arachidonic acid, but has excluded consideration of the second moiety formed stoichiometvically--lysophospholipids (lyso-PLs). Recently we reported (1-/4) that lysophosphatidylchotine, provided

exogenously, could


I release


a step

apparently distal to energy production, whereas lysophosphatidylethanolamine or -serine were essentially inactive (1-4).

We therefore proposed that lysophosphatidylcholine, as

an endogenous cationic amphiphile, could mobilize a membrane-associated Ca ++ pool, allowing its translocation to the exocytotic machinery.

This formulation was supported

by the identical results seen with two maneuvers designed to augment the accumulation of endogenous lysophosphotipids--provision of exogenous PLA 2 (4,5), or o f E-hydroxy-

A b b r e v i a t i o n s : L y s o - P l , l y s o p h o s p h a t i d y l i n o s i t o l ; Lyso-PA, l y s o p h o s p h a t i d i c a c i d ; Lyso-PG, l y s o p h o s p h a t i d y l g l y c e r o l ; Lyso-CL, l y s o c a r d i o l i p i n ; Lyso-PL, l y s o p h o s p h o l i p i d ; PI, p h o s p h a t i d y l i n o s i t o l ; PLA, phospholipase A; I , i n s u l i n . 0006-291 X/86 $l.50 Copyright © 1986 by Academic Press, Inc. All rights (?f reproduction in any jbrm reserved.


Vol 138, No 2, 1986


mercuribenzoic acid, an agent which impedes the re-esterification of endogenous islet lyso-PLs with arachidonate (1-3). If this simple formulation is correct, anionic lysophospholipids might be inactive. To examine this hypothesis, we tested the effects on I release of several Iysa-PLs which could be formed from acidic phospholipids by the action of PLA 2 or PLA 1. One of these lysophosphatidic acids (lyso-PA), might be formed not only by PLA 1 or PLA 2 action on phosphatidic acid (6,7) or by the phosphorylation of monoacylglycerob but also via de nova synthesis in islets through the acylation of the 3-carbon phospholipid glycerol backbone derived directly from the metabolism of glucose (8).

Materials and Methods

Islet Isolation and Incubation. Intact rat islets from fed, male Sprague-Dawley rats were isolated after pancreatic digestion and concentration on a ficoIl gradient, as previously described in detail (2,4,5,9). The procedures used to assess insulin (I) release from intact rat islets during static, 30 rain incubations in Krebs Ringer-Bicarbonate buffer have also been described (2,4,5,9). Since our previous studies demonstrated that the effects of lysophospholipids on I release were markedly reduced by the presence of albumin, incubations containing lysophospholipids were carried out in 750pl of albuminfree medium, which was brought to a final concentration of 0.5% BSA (for adequate recovery and measurement of secreted insulin) by addition of 250#1 of 2% BSA at the end of the incubation. Stock solutions of lyso-PI (Avanti Polar Lipids; Birmingham~ Alabama) were made up in KRB buffer containing 0.1 N NaOH by vortexing. Stock solutions of PI (from soybean; Sigma) were made in chloroform. Lysophosphatidylglycerol (lyso-PG~ from from egg yolk lecithin) was from Sigma (St. Louis, MO), and mona- or di-lysocardiolipin (lyso-CL) was from Avanti; stock solutions were made in chloroform. Lyso-PA (from egg lecithin; purchased from Serdary; London, Ontario9 Canada) was supplied in chloroform. Small aliquots (~SpI) of the stock solution of lysoPI were added directly to l m l KRS medium to achieve the desired final concentrations of test compounds. In the case of the PI~ lyso-PA, lyso-PG or lyso-CL, the diluent was first evaporated under an argon stream, KRB buffer was then added, and the test compounds returned to solution via sonification or vortexing. Control tubes always contained identical dituents as used for the test chemicals. Test Iipids or potential inhibitors were generally present during the final 50 min incubation period only. However, 8-(N-N-diethylamino) octyl 3,4,5-trimethoxybenzoate HCL (TMB-8, Sigma) or antimycin A (Sigma) were present in both preincubation and incubation periods. For addition of LaCI 3 (Sigma)~ the routine KRB medium was replaced with a bicarbonate-, sulfate-, and phosphate-free medium (supplemented with 25ram HEPES buffer) in order to avoid any precipitation of the La +++ by the anions. For studies of the reversibility of lyso-PI, islets were removed on their mesh filters from the lyso-PI-supplemented medium after the first 50 min incubation period and were transferred into new tubes containing fresh KRB buffer with 1.7mM glucose and 0.5% bovine serum albumin, for 20 mins. Following this wash step, islets were transferred to new medium containing 1.7 or 16.7mM glucose in order to test the reversibility of the lyso-PI effect and to examine the effect of a prior exposure to lyso-PI (during the first incubation period) on the insulin response to glucose during the second incubation period. Measurefpe_nts a n d D a [ a A n a ~ s i s . Insulin was measured by radioimmunoassay using an antibody donated by Dr. O. Kolterman (San Diego, CA) and rat insulin standards (Nova Research Institute, Bagsvaerd, Denmark). Data for each experiment are presented as mean (g) + SEM of insulin secreted (pU/1O islets/30 mins) in replicate tubes from one given pool of islets. Insulin levels were analyzed by nan-paired t testing using only comparisons of replicates from the same pool of islets studied on the same day. 721

Vol. 138, No. 2, 1986


Results and Discussion

Lyso-PI led to a concentration-dependent and saturable rise in insulin (l) release at 1.Tram glucose with an apparent half-maximal response at 4g-SO#g/mr and maximum reached by <150~g/ml (Fig. l).

The threshold response for lyso-PIwas between 10 and

20pg/ml (Table 1). Lyso-P! (Z00#g/ml) did not alter the immunoassay for I. P[ itself had no effect on [ re[ease (Table t). The response to tyso-P[ involved physiologic (exocytotic) release mechanisms as opposed to a toxic (lytic) effect since (1) it was dose-dependent, saturable (Fig. 1) and reversible upon washing out tile compound (Table 2); (2) islets previously exposed to lyso-Pl and then washed responded normally to a physiologic stimulus (16.7mM glucose, Table 2); and (5) it was inhibitable, by nickel chloride (NiCi2, 2mM) or lanthanum chloride (LaCt 5, 2raM) (Table 1). The tatter observations were not reproduced by extracellular Ca ++ depletion plus 0.2mM EGTA or the inhibitor of intracelluiar Ca ++ mobilization TMB-8 (data not shown), using conditions and concentrations of the drugs under which the effect of 16.7mM glucose is inhibited (not shown). Thus the effects of LaCl 5 and NiCt 2 presumably reflect their respective actions to impede the mobilization of superficial membranous Ca ++ stores (1O) and tile intracellular effects of Ca ++ (11). The exocytotic nature of the release was further supported by the reduction

in secretion at reduced ambient temperature (Table 1)~ which impedes

physiologic release but actually increases the lyric, toxic effect of lyso-PLs (12).


effect of ;yso-PI was at least additive to, and probably was synergistic with, that of

400. *




p< .001

(6) ~ ; )


E o 300'




zj ,J

0 .--/,L r





16o 1go


[Lyso-PI], pg/ml

Figure 1, Insulin r e l e a s e (at 1.7ram glucose) induced by i n c r e a s i n g c o n c e n t r a t i o n s of lyso-PI.


Vol. 138, No. 2, 1986



E f f e c t o f Lyso-Pl or PI in the presence or abscence o f various p o t e n t i a l




EXPERIMENT 1 a. control (l.7mM glucose) b. lyso-PI, lOpg/ml c. lyso-PI, 20pg/ml d. lyso-PI, 25pg/ml

123_+22 137+20 219_+/4 234_+18

(3) (6) (6) (5)

EXPERIMENT 2 (anion-poor buffer) a. control (1.7mM glucose) b. lyso-PI, 5gpg/ml c. lyso-PI + LaC13, 2mM d. lyso-PI, 150pg/ml e. lyso-PI + LaCI3, 2raM

169_+26 279_+33 70+8 559_+35 72+_5

(5) (3) (5) (8) (6)

EXPERIMENT 3 a. control (1.7mM glucose) b. lyso-PI, 150pg/ml c. lyso-PI + NiCI2, "_#m!vl

100_+7 ~15_+47




EXPERIMENT/4 a. control (37°C, 1.7mM glucose) b. lyso-PI, 50pg/ml c. lyso-PI, 50pg/ml (16°C)

73_+6 (3) 277_+26 (4)

EXPERIMENT 5 a. control (1.7mM glucose) b. control + lyso-PI, 251jg/ml c. glucose, 16.7mM d. glucose, 16.7raM + lyso-PI, 25pg/ml EXPERIMENT 6 a. control (1.7ram glucose) b. Pl, 25pg/mI PI, 2501jg/m[

p < .00[ vs. a p<.01vs, a

p < .001 vs. (b) p < .001 vs. (d)




126_+2t 212+14 1401-+156 1598+89

(3) (3) (3) (3)

p<.O01 vs.(b)

p < .OOl vs. (b)

68_+10 (5) 65+_15 (3) 74_+7 (3)

* p U / 1 0 islets/30 mins; ~ +_ S-~M,_ (n) = number of replicate tubes/condition FJSA excluded from incubations

16.7mM glucose (Table 1).

However, the e f f e c t

of lyso-PI at 1.7ram glucose was

resistant to a n t i m y e i n A (data not shown) and thus i t presumably could p r o m o t e release distal to, or largely independently of, energy flux or m i t o c h o n d v i a l ATP production. M o r p h o l o g i c a l l y , lyso-PI ( 5 0 p g / m t ) - t r e a t e d islets appeared normal and excluded tvypan blue; at 1501~g/ml, l y s o - P I - t v e a t e d - i s l e t s remained essentially normal in appearance, e x c e p t f o r a f e w swollen, although apparently intact~ ceils at the periphery of the islet. Lysophosphatidylglycerol (lyso-PG)

also led to


dose-responsive increases



Vol 138, No 2, 1986 TABLE 2:


Reversibility of lyso-Pi-induced insulin release (at 1.7mM glucose) and tack of effect on the subsequent glucose (16,7mM) responsivity of the islet Insulin* (lst Incubation) --~ Wash ~

Insulin* (2nd Incubation)




glucose, 1.7mM

95+12 (7)

glucose, Z.7mM + lyso-PI, 50tJg/ml

245+_17 (8)

~-JG' i.7mM Q, 16.7mM E G'

glucose, !.7mM + lyso-PI, 150l~g/mI

483+40 (8)


769+-44 (4) 44+3





G, 16.7m~4


59+_17 (4)

G, 1.7raM

67_+11 (4)

G, 16.7mM

811-+62 (4)

*insulin expi"essed as pU/10 islets/30 mins ()Z -+ SEM) (n) = number of replicates/condition

release at 1.7ram glucose, with a minimal effective concentration of < 20pg/mt (Table 3). However, unlike that of tyso-P[, the effect of lyso-PG did not appear to be saturable (at least up to 250~tg/ml, the highest concentration tested).

Lyso-PG also was additive

to, if not truly synergistic with, I release induced by 16.7mM glucose (Table 3).


effect of lyso-PG (like tyso-PI) was inhibited by NiCI 2 (-89+4%, n=4, p < .001) or reduced ambient temperature (16°C = -65_+8%, n=5, p <.001), but not by antimycin A. Lyso-PA, in contrast, had absolutely no effect at 1.TmM glucose (at 0.25, 1, 2, 5, 10, 20~ 50, 125, 250pg/ml) or at 5.5 or 16.TmM glucose (at 5, 50, o r 2 0 0 p g / m l ) .


(2 or 5Opg/m[) also could not reverse the inhibition of glucose (16.7mM)-induced I release caused by the inhibition of glucose metabolism caused by 16.7mM mannoheptulose (data not shown).

Thus one can reasonably exclude the possibility that lyso-P[ stimulated [

release by cleavage to lyso-PA through the action of a phosphotipase D (13). The validity of these studies is based on the assumption that exogenously provided tyso-PA has access to potential sites of action, although endogenous tyso-PA is synthesized intraceJlularly. This assumption seems reasonable since, at least in platetets, lyso-PA has an extracellular site of action (14) and exogenously-provided lyso-PA activates the cells (14-17). Exogenously-provided lyso-PA could also overcome the inhibition of thrombin-induced platelet responses by the phosplnolipase inhibitor mepacrine (18), but could not, in the current study, reverse the inhibition of glucose-induced I release caused by the PLA 2 inhibitors, mepacrine (100pM) or bromphenacyl bromide (25pM) (data not shown). 724

V o l 138, No. 2, 1986


TABLE 3: Effect of Lyso-PG and Lyso-CL on Insulin Release at 1.7ram Glucose CONDITION

Experiment One a. glucose (G), 1.7mM b. G 1.7mM + lyso-PG, G 1.7mM + lyso-PQ, G 1.7mM + lyso-PG, G 1.7ram + lyso-PG, G Z.7mM + lyso-PG, G $.7mM + lyso-PG, G 1.7mM + lyso-PG, G 1.7mM + lyso-PG,


2138_+33 (3) 13.5 pg/ml 2 pg/ml 10 tJg/ml 20 50 1Jg/ml 100 lag/ml 150 pg/ml 250 iag/ml

Experiment Two a. glucose, 1.7ram b. glucose,l.7mM + Lyso-PG, 25 lag/rot



164_+26 129_+48 279_+29 535_+70 837+_135 1291+92 2022_+233

(3) (3) (/4) (5) (5) (3) (3)

63+4 (2) 290_+77 (3)


glucose, 1 6 . 7 m M



glucose, 16.7ram + Lyso-PG, 25pg/ml

1539+162 (3)

Experiment Three a. glucose, 1.7mM b. glucose, t.7mM + monolyso-CL, $pg/ml glucose + monotyso-CL, 20pg/ml glucose + monolyso-CL, 1001ag/ml c. glucose, 1.7mM + dilyso-CL, llag/ml glucose + dilyso-CL, 201ag/ml glucose + dilyso-CL, 100ljg/ml

53+6 /44_+10 117_+5 101+9 29_+3 79+4 439+26


(3) (3) (3)--p < .Ol vs. (a) (3)--p < .02 vs. (a)

(3) (3)--p < .05 vs. (a)

(3)--p < .00 t--vs. (a)

*pU/10 islets/30 rains 0Z + SEM) (n) = number of replicates/condition

Furthermore, we have observed modest stimutatory effects of mono- and especially of di-lysocardiolipin (lyso-CL) on I release (Table 3) even though cardielipin synthesis is a l m o s t c o m p l e t e l y i n t r a c e l l u l a r , in m i t o c h o n d r i a (19).

Thus, l y s o - P L - i n d u c e d I r e l e a s e

seems to have considerable true structural specificity, with only the lyso-derivatives of PC, PQ, PI and CL being active whereas lyso-PE, lyso-PS, lyso-PA, intact Pl_s and glycerophosphoryl-base derivatives are inactive (1-4).

However, i t is still possible that

lyso-PA may serve an indirect ( i . e , structural or phospholipid precursor) role in glucoseinduced I release and that such a role can not be reproduced by s h o r t - t e r m exogenous provision. Anionic lysophospholipids seemed capable of both i n i t i a t i n g I release at 1.7mM glucose and synergizing with 16.7mM glucose.

The mechanism of action of lyso-PI or

lyso-PG to augment secretion is not c l a r i f i e d by these studies.

However, the anionic

nature of these lyso-PLs (in contrast to our previous results with the cationic lysophosphatidytchotines; refs. 1-/4) could be taken to suggest that simple electrostatic displacement of membrane-bound Ca ++ may not be a satisfactory explanation. 725

On the other

Vol 138, No 2, 1986


hand, lyso-PI has been shown capable of releasing Ca ++ from mitochondria (20).

isolated rat


Also, it is possible that exogenous l y s o - P l s could s a t u r a t e the r e -

esterification mechanism for endogenous lyso-PLs or act as lyso-acceptors in transacylation reactions.

The result in either case might be the accumulation secondarily of

lysophosphatidylcholine, or other endogen£us lyso-compounds.

In this context, it should

be appreciated that, while the concentrations of lyso-PLs used may seem high at first glance, the translocation of lysophospholipids across intact membrane bilayers is slow compared to the 30 minute incubations studied here (21), and thus only a small percentage of the lyso-PI may have reached the inner leaflet, where i t could mimic the amounts generated by the endogenous phospholipase A 2.

Increased fusigenicity of

biologic membranes caused by lysophospholipids (22) or fission of secretory granules (25) could also be involved. Further studies w i l l be needed to resolve these questions. It is likely that lyso-PI and lyso-PG can be formed in the islets through the action of phospholipase A or through transacylation reactions (24).

Several authors have

reported the presence of PLA2-like activity in homogenates of rat islets (25,26) or intact rat islets (5).

Furthermore, Schrey and Montague have reported that guinea pig islet

homogenates can form lyso-PI via a phospholipase of the A 1 type (27). The presence in the pancreatic islet of lyso-PI and lyso-PG is also suggested by the incorporation of exogenous araehidonic acid into PI and PG (28,29).

Lyso-PI could potentially also be

formed via the successive dephosphorylation of lysopolyphosphoinositides (30).


tatter are unique among lysophospholipids in their resistance to acylation and to removal from the membrane, and in their ability to form cation channels or lipid membranes; refs. 30,31.

It might t h e r e f o r e be of interest to t e s t their e f f e c t s on e x o c y t o s i s i n future


Acknowledgments Funded by the Veterans Administration, of which Dr. Metz is a Clinical Investigator. The expert technical assistance of Ms. Mary Rabaglia and Mr. Doug Holmes and the helpful and stimulating discussions with Dr. Eric Brass are gratefully acknowledged.

References 1. Z. 3.

Metz, S., (1986) Proc. X[! Cong. Intl. [Diabetes Fed., Excerpta Medico, Elsevier Scientific Publishers, The Netherlands, in press. Metz, S., (i986) J. Pharmaco[. Exp. Ther., in press. Metz, S. (1986) Clin. Res. 34, 62a. 726

Vol 138. No 2, 1986 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 1/4. 15. 16. 17. 18. 19. 28. 21. 22. 23. 2/4. 25. 26. 27. 28. 29. 30. 31.


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