Production of inositol trisphosphates and inositol tetrakisphosphate in stimulated pancreatic islets

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Biochimica et Biophysica Acta 927 (1987) 112 116 Elsevier

BBA 11644

Production of inositol trisphosphates and inositol tetrakisphosphate in s t i m u l a t e d p a n c r e a t i c islets L. B e s t

a,

S. T o m l i n s o n

a,

P.T. Hawkins b and C.P.Downes

b

" Department of Medicine, University of Manchester, Manchester and ~ Department of Cellular Pharmacology, Smith, Kh'ne & French Research Ltd., Welwyn ( U.K.) (Received 21 July 1986)

Key words: Inositol phosphate metabolism: Glucose stimulation; Carbamylcholine: (Rat pancreatic islet)

Glucose and carbamylcholine caused concentration-dependent increases in the production of total [3H]inositol phosphates in [3H]inositol-labeiled rat pancreatic islets. When extracts from islets stimulated with glucose, carbamylcholine or depolarising concentrations of K + were analysed using anion-exchange high performance liquid chromatography, increased production of [3H]Insl,4,5-P3 was detected, and in addition, elevated levels of two other labelled compounds which co-chromatographed with Insl,3,4-P3 and Insl,3,4,5-P4. In the case of carbamylcholine and high K +, such an effect was apparent within 20 s, whereas glucose appeared to cause a delayed response. In the presence of 5 mM LiCI, the accumulation of Insl,3,4-P3 was more marked. The presence of LiCl had no major influence on the levels of Insl,4,5-P3 or Insl,3,4,5-P4. It is suggested that the stimulation of pancreatic islets with glucose, carbamylcholine or high K + results in the hydrolysis of inositol lipids with the production of Insl,4,5-P3 and in addition, Insl,3,4-P3 and Insl,3,4,5-P4. The physiological functions of these novel inositol phosphates in islets remain to be established.

Introduction The stimulation of pancreatic islets with nutrients (e.g., glucose) or certain neurotransmitters (e.g., carbamylcholine) results in the hydrolysis of inositol phospholipids [1,2] and the formation of inositol phosphates, including InsP 3 [2,3]. It has been proposed that Insl,4,5-P~ has a physiological

Abbreviations: PtdIns, phosphatidylinositol; PtdIns4,5-P2, phosphatidylinositol 4,5-bisphosphate; InsP, inositol monophosphate: Insl-P, inositol I-phosphate; InsP2, inositol bisphosphate; Insl,4-P2, inositol 1,4-bisphosphate; InsP 3, inositol trisphosphate, Insl,4,5-P3, inositol 1,4,5-trisphosphate; Insl,3,4-~. inositol 1,3,4-trisphosphate; InsP4, inositol tetrakisphosphate; Insl,3,4,5-P4, inositol 1,3,4,5-tetrakisphosphate. Correspondence: Dr. L. Best, Department of Medicine, University of Manchester, Oxford Road, Manchester, M13 9PT, U.K

role in mobilising Ca 2+ from intracellular stores [4], most likely endoplasmic reticulum, and it has subsequently been shown that this substance can release Ca 2+ from permeabilised insulin-secreting cells [5] and from microsomes prepared from rat insulinoma cells [6]. The lipid product of inositol lipid breakdown, diacylglycerol, has been shown to cause protein phosphorylation by activating protein kinase C [7], an enzyme whose activity has been demonstrated in islet homogenates [8]. Thus, it is likely that the hydrolysis of inositol lipids in stimulated islets results in the generation of two distinct intracellular signals which modulate the secretion of insulin. Recent studies of inositol lipid metabolism in other tissues have revealed the formation of two novel inositol phosphates, Insl,3,4-P 3 and Insl,3,4,5-P 4 [9-11]. Both of these compounds are thought to arise indirectly from receptor-stimu-

0167-4889/87/$03.50 ;C 1987 Elsevier Science Publishers B.V. (Biomedical Division)

113 lated release of Insl,4,5-P 3 from PtdIns4,5-P2, Insl,3,4,5-P 4 can be formed by the action of an Insl,4,5-P 3 kinase [12,13] and Insl,3,4-P 3 subsequently formed by a 5-phosphate-specific hydrolysis of the Insl,3,4,5-P 4 [11,13]. We have investigated the formation of Insl,4,5P3, Insl,3,4-P3 and I n s P 4 in pancreatic islets challenged with glucose, carbamylcholine and depolarising concentrations of K+; three stimuli known to trigger the breakdown of inositol lipids in islets. Materials and Methods Pancreatic islets were isolated from fed adult rats by collagenase digestion [14]. In experiments designed to measure total production of inositol phosphates, batches of 100 islets were preincubated in 1.0 ml gassed bicarbonate medium [15] containing 2.8 m M glucose, 0.25% bovine serum albumin and 6 #Ci myo-[3H]inositol (Amersham; 12-15 C i / m m o l ) for 2 h at 37°C. The medium was then removed, the islets were washed with 5 ml unlabelled medium and finally placed in 0.9 ml medium containing 5 m M LiC1 and 1 m M unlabelled myo-inositol. Incubations were started by the addition of 0.1 ml medium containing appropriate concentrations of either glucose or carbamylcholine. The islets were incubated for a further 20 min and incubations terminated by the addition of 1.0 ml ice-cold trichloroacetic acid and 0.1 ml 100 m M EDTA. The tubes were then centrifuged to precipitate proteins and the supernatant removed and extracted with 5 vol. water-saturated diethyl ether. The samples were then mixed with 4 ml water and applied to a column containing 1 ml Dowex AG1X8 (200-400 mesh), formate form. The columns were washed with 15 ml water and total inositol phosphates were eluted with 3 ml 1 M a m m o n i u m f o r m a t e / 0 . 1 M formic acid. The radioactivity in the samples was counted following the addition of 10 ml Aquasol scintillant. In experiments designed to investigate the production of individual inositol phosphates, batches of 200 islets were treated as described above, with the exception that LiC1 was sometimes omitted from the incubation medium (see Table I). Inositol phosphate sin the islet extracts were re-

TABLE I PRODUCTION OF [3H]Ins1,4,5-P3, [3H]Insl,3,4-P3 AND [3H]Insl,3,4,5-P4 IN BATCHES OF 200 ISLETS IN RESPONSE TO GLUCOSE, CARBAMYLCHOLINE(CCH) OR HIGH K + The figures represent determinations from two individual experiments and show percentage of control values (see Results).

A. No LiC1 Glucose (20 mM) 20 s 5 min CCH (i0 5 M) 20 s 5 min K + (120 mM) 20 s 5 min

Ins1,4,5-P3 Insl,3,4-P3

Insl,3,4,5-P4

79, 96 138, 215

62, 68 202, 180

96, 179 221,391

462,243 149, 195

485, 766 353,453

742, 368 297, 284

114, 144 151,172

457, 357 262, 305 6 4 3 , 6 4 2 316,295

B. 5 mM LiC1 Glucose (20 mM) 5 min 135, 161 CCH (10 5 M) 5 min 282, 178 K ÷ (120 mM) 5 min 332, 207

839, 1017 543,370 10 846, 4891 664,531 16 455, 10 155 132, 122

solved by anion-exchange high-performance liquid chromatography [9] using a 0.46 x 25 cm column packed with Partisil 10 SAX (Technicol, Stockport, U.K.). Half of the sample was spiked with 32p-labelled Insl,4,5-P 3 and injected onto the column, which was run at a flow rate of 1.25 m l / m i n using a non-linear gradient from 0 to 1.7 M ammonium formate buffered at p H 3.7 with phosphoric acid [11,13]. 0.25-ml fractions were collected and mixed with 1.0 ml methanol and 4 ml scintillarit, and their radioactivity was determined. [32p]Insl,4,5-P3 and [32p]Insl,4-Pa were prepared by phospholipase C-catalysed hydrolysis of 32p-labelled erythrocyte membranes [16]. L-Insl-P was purchased from Amersham International, U.K., and carbamylcholine from Sigma.

Results Glucose and carbamylcholine stimulated the production of [3H]inositol phosphates in [3H]inositol-labelled islets, as previously reported [2,3]. The concentration-response curves for total [3H]-

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Fig. 1. Total [3 H]inositol phosphate production in isolated rat islets in response to carbamylcholine and glucose. Each point represents the mean of duplicate determinations.

inositol phosphates accumulated during 20 rain in the presence of either of these two agonists and 5 mM LiC1 are shown in Fig. 1. LiC1 was included in these experiments in order to block inositol phosphate hydrolysis and thus obtain a more accurate measure of the activity of phospholipase C [17]. Carbamylcholine was the more effective stimulus at maximal concentrations. When the [3H]inositol phosphates extracted from 200 islets were analysed by anion-exchange HPLC, a number of compounds were resolved (Fig. 2). [3H]Insl,4,5-P3, [3H]InsP2 and [3H]InsP were identified by cochromatography with the ap-

propriate 32p-labelled internal standard (data not shown). Two further 3H-labelled compounds were formed which had the precise mobilities on this chromatographic system of Insl,3,4-P 3 and Insl, 3,4,5-P4 ([13]; Fig. 2). The values for [3H]Insl,4,5P3, [3H]Insl,3,4-P3 and [3H]Insl,3,4,5-P4 in unstimulated islets were 602 + 20, 396 + 42 and 388 + 32, respectively. In the presence of 5 mM LiC1, the corresponding values were 1346 +_ 257, 116 _+ 16 and 372 _+ 51. The changes in the levels of [3H]Insl,4,5-P3, [3H]Ins1,3,4-P3 and [3H]InsP4 in [3H]inositollabelled islets stimulated with glucose (20 mM), carbamylcholine (10 5 M) or high K ÷ (120 mM) are shown in Table I. In the case of glucose, no increases were apparent after 20 s, although the levels of all three inositol phosphates were raised after 5 min of stimulation. In contrast, the effects of carbamylcholine were more rapid, the levels of [3H]Insl,4,5-P3 [3H]Ins1,3,4-P3 and [3H]Insl,3,4, 5-P4 being elevated within 20 s of stimulation and remaining raised after 5 min. High K + also increased the production of these three inositol phosphates after 5 min of stimulation. The stimulation of islets in the presence of 5 mM LiC1 with glucose, carbamylcholine or high K ÷ resulted in alterations in the proportions of accumulated [3H]inositol polyphosphates (Table I). Li ÷ exerted very selective effects on the levels of the inositol polyphosphates examined. Thus, it greatly potentiated Insl,3,4-P 3 accumulation, particularly when the stimulus was carbamylcholine

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or high K +, yet did not influence the accumulation of Insl,4,5-P 3.

Discussion The concentration-dependence of inositol phosphate formation in pancreatic islets in response to glucose and carbamylcholine reported here closely resembles those found in earlier studies measuring 32p-labelling of inositol lipids [18,19], which is a more indirect index of stimulated phospholipase C activity than the measurement of inositol phosphates. More recent studies have demonstrated the formation of InsP 3 in islets in response to nutrients and neurotransmitters [2,3]. The present work has demonstrated that, as in stimulated brain [11], parotid [9], liver [20,21], DMSO-differentiated HL-60 cells [21] and exocrine pancreas [21], this InsP 3 consists of two isomers, namely Insl,4,5-P 3 and Insl,3,4-P 3. Furthermore, we have demonstrated stimulated formation of a compound with the chromatographic properties of Insl,3,4,5-P 4. Glucose, carbamylcholine and high K ÷, three distinct types of agonist, each stimulated the formation of Insl,3,4-P 3, Insl,4,5-P 3 and Insl,3,4,5P4, suggesting that each triggers inositol lipid metabolism in a fundamentally similar way. Since in both brain and parotid, Insl,3,4-P 3 and Insl,3,4,5-P4 can be derived from Insl,4,5-P 3 [11,13], the simplest explanation is that, in each case, all three inositol phosphates are formed by initial activation of Ptdlns4,5-P2-specific phospholipase C. This hypothesis requires confirmation that Insl,4,5-P 3 kinase and Insl,3,4,5-P4 5phosphatase activities exist in pancreatic islets. The ability of muscarinic receptors to couple to PLC, possibly via a guanine nucleotide regulatory protein [22], is well documented. It is not yet known, however, how glucose might activated this enzyme in pancreatic islets. One possibility is that stimulated inositol lipid breakdown may occur in this tissue as a result of Ca 2÷ entry into islet cells [23], which is known to occur during glucose stimulation [24]. Consistent with this idea is the ability of depolarising concentrations of K ÷ to induce inositol lipid breakdown in islets (Ref. 23 and present results). At concentrations of glucose and carbamylcholine which induced a similar extent of accumulation of total inositol phosphates

during 20 min in the presence of LiC1, the formation of Insl,4,5-P 3, Insl,3,4-P 3 and Insl,3,4,5-P 4 in response to glucose was slower than the response to carbamylcholine. We have previously observed a delay in glucose-stimulated inositol lipid metabolism [1], and although the reasons for this lag are unclear, it is possible that transport a n d / o r metabolism of the sugar are necessary prior to activation of phospholipase C. Li + (5 raM) caused a very large, selective potentiation of stimulated Insl,3,4-P 3 accumulation. This effect of Li + upon Insl,3,4-P 3 levels is far greater than that previously observed in caerulein-stimulated exocrine pancreas [21]. The effect of Li + is most easily explained by a potent inhibition of Insl,3,4-P 3 phosphatase, though, as far as we are aware, no studies have yet been published on the properties of this enzyme in any tissue. The lack of effect of Li + on Insl,4,5-P 3 levels is entirely consistent with previous observations that Insl,4,5-P3-phosphatase is Li+-insensi tive [25,26]. The demonstration that inositol phosphate formation in islets stimulated with glucose, carbamylcholine or high K + is remarkably similar to that seen in brain and parotid gland stimulated with carbamylcholine [11,13] and hepatocytes stimulated with vasopressin [20] suggests that common pathways of inositol phosphate formation and degradation may apply to a wide variety of cell types and stimuli. It also supports the idea that Insl,3,4,5-P 4, which apepars to be rapidly formed and degraded in those tissues where it has been studied [11,13], may have some ubiquitous biological role in the response analogous to, but distinct from Insl,4,5-P3-induced Ca 2+ release.

Acknowledgement This work was supported in part by the Medical Research Council.

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