The effect of monooleoylglycerol on insulin secretion from isolated perifused rat islets

Diabetologia, Jun 1989

W. S. Zawalich, K. C. Zawalich, H. Rasmussen

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The effect of monooleoylglycerol on insulin secretion from isolated perifused rat islets

Diabetologia The effect o f monooleoylglycerol on insulin secretion from isolated perifused rat islets W. S. Zawalich 0 1 K. C. Zawalich 0 1 H. Rasmussen 0 1 0 1YaleUniversitySchoolof Nursing,and 2Departmentof InternalMedicineand CellBiology,YaleUniversitySchoolof Medicine , New Haven,Conn. , USA 1 Dr. W. S.Zawalich Yale University School of Nursing P. O. Box 9740 885 Howard Avenue New Haven, Conn. 06536-0740 USA Summary. The effect of monooleoylglycerol on cholecystokinin- and tolbutamide-induced insulin secretion was examined in isolated perifused rat islets. In the presence of 5.5 mmol/1 glucose, addition of 10 nmol/1 cholecystokinin or 50 p~mol/1tolbutamide had practically no effect on insulin secretion. Combined tolbutamide and cholecystokinin led to a biphasic insulin secretory response which was significantly enhanced by addition of 50 ~tmol/1 monooleoylglycerol, an inhibitor of diacylglycerol kinase. Monooleoylglycerol (50 p.mol/1)alone had a minimal stimulatory effect on insulin release in the presence of 5.5 retool/1 glucose. Perifusion of islets with 1 lxmol/1forskolin had no significant effect on basal insulin secretion in the presence of 5.5 mmol/1 glucose, but Insulin secretion; diacylglycerol; isolated islets; second messengers; phosphoinositides - 9 Springer-Verlag1989 There is considerable evidence which supports the view that protein kinase C plays a role in mediating insulin secretion from the B cell of the pancreas [ 1-3 ]. However, there is no consensus as to its precise and obligatory involvement in this process [ 1-5 ]. In previous studies in perifused rat islets, we showed that tolbutamide and forskolin acted synergistically with 12-0-tetradecanoyl phorbol-13-acetate (TPA) to stimulate insulin secretion [ 6, 7 ] and proposed that protein kinase C plays a key role in mediating the sustained or second phase of glucose-induced insulin secretion [ 6-8 ]. However, this combination of pharmacologic agents did not, even at optimal concentrations, induce as great an insulin secretory response as did 16.7 mmol/1 glucose, the pre-eminent physiological agonist for stimulated insulin secretion. Our recent studies [ 9-12 ] of the complex relationships between the actions of cholecystokinin 8-sulfate (CCK-8S) and of glucose on insulin secretion have led to further insights into the mechanisms by which insulin secretion is controlled. In these studies, it was shown that 100-200 nmol/1 CCK-8S induces a rapid hydrolysis of polyphosphoinositides, the production o f inositol phosphates, and a sustained increase in efflux of markedly enhanced the responses to both cholecystokinin plus tolbutamide, and to the combination of cholecystokinin, tolbutamide and monooleoylglycerol. Lowering the glucose level to 2.75 mmol/l abolished the profound stimulatory effect to these agonist combinations on insulin release. Finally, monooleoylglycerol also enhanced the first and second phase insulin secretory responses induced by 20 mmol/1 glucose. These results are discussed in relationship to the possible role of protein kinase C in mediating insulin secretion. [3H]-inositol from islets in which the phosphoinositide pool was prelabelled. The effect of CCK-8S on these parameters of cellular activation is nearly identical whether islets are perifused with 2.75 or 7.0 mmol/1 glucose, but the accompanying insulin secretory response is quite different; CCK-8S induces a biphasic insulin secretory response from islets incubated in media containing 7 mmol/1 glucose, but there is no significant secretory response from islets incubated in 2.75 mmol/1 glucose. Pharmacologic agents were used to determine whether or not either Ca 2+ influx a n d / o r an increase in cAMP concentration were sufficient to restore the insulin secretory responsiveness of islets (incubated in 2.75 mmol/1 glucose) to CCK-8S. Forskolin, an activator of adenylate cyclase, restores partial responsiveness to CCK-8S and tolbutamide, a stimulator of Ca 2+ influx, further enhances the restorative effect of forskolin [11]. However, addition of CCK-8S to islets incubated in 2.75 mmol/1 glucose and treated with either 1 ~tmol/1 forskolin a n d / o r 200 txmol/1 tolbutamide, leads to a marked first phase insulin secretory response, but no sustained second phase [ 11 ]. These results imply that glucose has an additional effect on islet cell metabolism related to the second or sustained phase of insulin secretion. We postulated that this is on the metabolism of diacylglycerol (DAG) by a pathway other than PI hydrolysis (see also [ 13 ]). This unique effect of glucose on D G production has recently been demonstrated [ 14 ]. Recently Bishop et at. [ 15 ] have reported that monooleoylglycerol (MOG) is a relatively specific inhibitor of the enzyme D A G kinase which converts D A G to phosphatidic acid. We have shown that M O G causes an increase in the D A G content of cultured 3T3 cells and that this leads to an activation of protein kinase C in these cells [ 16 ]. The present study reports the effect o f monooleoylglycerol on glucose- and CCK-8Sinduced insulin secretion under several circumstances. We reasoned that if glucose and CCK-8S have their postulated effects on D A G metabolism then application of M O G might significantly enhance both CCK8S- and glucose-induced insulin secretion. Materials and methods The detailed methodology employed to assess insulin output from collagenase isolated islets has been previously described [ 9-12 ]. Male Sprague-Dawley rats purchased from Charles River (Kingston, NY, USA) were used in all studies. The animals were fed ad lib and at the time killed weighed between 300-400g. After Nembutal-induced (50 mg/kg) anesthesia, islets were isolated by collagenase digestion and perifused to assess secretory responsiveness. Groups of 12-15 islets were loaded onto nylon filters and perifused at a flow rate of 1 ml/min for 30 rain to establish stable, basal secretory rates. After this stabilisation period, the islets were then subjected to various protocols indicated in the Figures. Effluent samples were collected and analysed for insulin content using rat insulin as standard (Lilly, Indianapolis, Ind., USA, Lot # 615-D63-12-3). Reagents Hank's solution was used for the islet isolation. The perifusion medium consisted of 115 mmol/1 NaC1, 5 mmol/l KC1, 2.2mmol/1 CaC12, I retool/1 MgC12, 24 mmol/1 NaHCO3, and 0.17 g/dl bovine serum albumin. Other compounds were added as indicated and the solution was gassed with a mixture of 95% 02/5% CO2. The t25I-insulin used for the insulin assay was purchased from New England Nuclear, Boston, Mass., USA. Bovine serum albumin (RIA grade), monooleoylglycerol (monoolein), sulfated cholecystokinin (fragment 26-33 amide), as well as the salts used to make the Hank's solution and perifusion medium were purchased from Sigma Chemical Company, St.Louis, Mo., USA. Forskolin was purchased from Calbiochem, La Jolla, Calif., USA. Tolbutamide (sodium salt) was the generous gift of Upjohn (Kalamazoo, Mich., USA). In the experiments involving MOG, DMSO was used as the solvent. The level of DMSO used (never greater than 0.2%) had no effect on insulin secretory patterns. Statistical Analysis Statistical significance was determined using the Student's t-test for unpaired data and where appropriate, analysis of variance. A p value less than 0.05 was taken as significant. Values presented in the figures or results sections represent means + SEM of the specified number of observations. Results CCK-8S (10 nmol/1) had a minimal and transient effect on insulin secretion from islets incubated in media containing 5.5 mmol/1 glucose (Fig. 1, left). Likewise, addition of 50 lxmol/1 tolbutamide causes only a very small and transient increase in insulin secretion (Fig. 1, center). No sustained insulin stimulatory effect of either compound is observed if the glucose level is 2.75 mmol/l. When CCK-8S and tolbutamide are combined, however, they induce a modest biphasic insulin secretory response from islets incubated in 5.5 but not 2.75 mmol/1 glucose (Fig. 1, right). I f islets (incubated in 5.5 mmol/1 glucose) are exposed to 50 ~tmol/1 MOG, along with CCK-8S and tolbutamide, there is a dramatic biphasic insulin secretory response (Fig.2, left) which is markedly enhanced when compared to CCK8S and tolbutamide alone (see Fig. 1, right). This insulin secretory response to the combination of MOG, CCK8S, and tolbutamide is similar to that induced by 20 mmol/1 glucose alone (Fig.2, right). It is noteworthy that 50 ~mol/l M O G alone has o13@ a minimal effect on insulin secretion in either the absence (Fig. 2, left) or presence of forskolin (Fig. 2, center). The most striking result is that found when islets are perifused with 5.5 mmol/1 glucose plus 1 p~mol/1 forskolin and then exposed to either 10 nmol/1 CCK-8S and 50 Ixmol/l tolbutamide, or to CCK-8S, tolbutamide and 50 [xmol/1 M O G (Fig. 2, center). The major effect of forskolin on the response induced by CCK-8S and tolbutamide is on the first phase o f insulin secretion. Peak first phase release is 145 _+12 pg. islet -1- rain -1 in the absence of forskolin and 1466 _+133 pg -islet -1. min - 1in the presence of forskolin. The rate of secretion at min 60 is 504 + 73 pg. islet, min (mean + SEM, n -- 4) without forskolin and 600+39 pg. islet - t .min -1 with forskolin. The further addition of M O G (50 gmol/1) along with CCK-8S, tolbutamide and forskolin (Fig. 2, center) leads to a marked enhancement of second phase secretion (2306+98 vs 1238+119 p g . i s l e t - l . m i n -1 in the presence and absence of forskolin, respectively). A small additional effect on the peak of the first phase secretion (1825+_191 vs 1466+ 133 pg-islet - l - r a i n -1 in the presence and absence of forskolin, respectively) was also observed. Under these conditions (CCK-8S, forskolin, tolbutamide, and MOG) both phases of the insulin secretory response are considerably greater than that induced by 20 mmol/1 glucose alone-peak first phase to this combination averaged 1825+191 vs 445 +_51 p g . i s l e t - l - min-1 for 2.0 mmol/1 glucose alone and peak second phase release to this combination averaged 2306 +_98 p g . i s l e t - t. min-1 vs 1283 +_116 pg-islet -1.rain -~ for 20 mmol/1 glucose alone. However, in the simultaneous presence of 50 Ixmol/1 MOG, 20 m m o l / l glucose does induce a second phase secretory response (1843 +_78 pg -islet - t . min -1) similar to that noted with the combination of 5.5 mmol/1 glucose, 50 ~mol/1 tolbutamide, 1 ~tmol/1 -B -C F i g t Influence ofcholecystokinin (CCK) and tolbutamide (TOL) on insulin secretory patterns in the presence of different glucose (G) concentrations. Groups of 12-15 islets were perifused for 60 rain with 2.75 ( H ) or 5.5 m m o l / l ( O ~ O ) glucose. For the final 30 rain, A) 10 n m o l / l CCK-8S (left panel) or B) 50 l%mol/l tolbutamide alone (middle panel) or C) in combination (right panel) were included in the medium. At least 4experiments were performed under each experimental setting. Mean values plus or minus selected SEM are given. This and all subsequent figures have been corrected for the dead space in the perifusion system, approximately 2.5 ml or 2.5 rain at a flow rate of 1 ml/min .--. "'~== 500 A 400 200 ~ 300 .E lOO Fig.2. Influence of forskolin (1 p~mol/l) on stimulated insulin secretion. Groups of 12-15 islets were perifused for 60 rain in the presence (middle panel) or absence (left and right panels) of 1 p~mol/1 forskolin. Left panel. A) Insulin output in the presence of 5.5 mmol/1 glucose plus the further addition of 50 p~mol/1 monooleoylglycerol (MOG) alone H , or the combination of 10 nmol/1 CCK-8S plus 50 ~tmol/1 tolbutamide (O-----O), or the combination of all 3 agonists (MOG, CCK-8S plus tolbutamide O - - - O ) is shown. Note that the line labelled ~ in Figure 1 C and @----@ in Figure 2A are the same data. Middle panel. B) Experimental protocol was identical to that outlined in the left panel except that 1 pmol/1 forskolin was present for the entire 60 rain. Right panel. C) Insulin output in response to a maximally effective glucose stimulus (20 mmol/1) plus or minus 50 Ilmol/1 MOG is shown. At least 4 experiments were performed under each experimental setting. Mean values _+ selected SEM are given. * = MOG: **CCK-8S plus tolbutamide; *** MOG, CCK-8S, and tolbutamide forskolin, 10 nmol/1 CCK, and 50 ~tmol/1 M O G (Fig. 2, center). Peak first phase insulin secretion rates in the presence o f 50 p~mol/1 M O G plus 20 mmol/1 glucose (953 + 98 pg. islet- 1. rain- t) were significantly greater than those noted with 2 0 m m o l / 1 glucose alone (445 +_51 pg. islet -a- rain- 1). Although not shown, the response to the agonist combination o f i p~mol/1 forskolin, 50 p~mol/1 tolbut W.S. Zawalich et al.: Second Messengers and Insulin Release IO00 ,T ~ amide, 10 nmol/1 CCK-8S, and 50 gmol/1 M O G was dependent, to a large extent, on the ambient glucose level. For example, at 2.75 mmol/1 glucose peak first phase insulin release to this agonist combination averaged 111_+ 8 pg.islet - 1 - m i n - t while release 30 rain after the onset o f stimulation was 74 +_3 pg. islet- 1. rain - t (n =3). In contrast, at 5.5 mmol/1 glucose these responses to the same combination n o w averaged 1825 + 191 pg. islet- 1 rain- 1 and 2306 + 98 p g . islet- 1 9min - t (n = 4 ) , respectively. Insulin release from the B cell provoked by the combination o f forskolin, tol butamide and CCK-8S was also entirely reversible. Removal o f these agonists resulted in basal insulin secretory rates within 20 rain (results not shown). Finally, it should be emphasised that the effects o f M O G are both reversible and dependent on calcium in flux (Fig. 3). The presence of the calcium channel antagonist nitrendipine (0.5 p~mol/1) virtually obliterated secretion in response to 20 mmol/1 glucose plus MOG. Despite being exposed to MOG, these islets were able, however, to respond to a subsequent exposure to 20 mmol/1 glucose. Discussion When the glucose concentration is 5.5 mmol/1, addition of 10 nmol/1 CCK-8S or a low dose o f tolbutamide alone does not have an appreciable effect on insulin secretion (Fig. 1). However, the combination of the two agents induces a biphasic insulin secretory response (Fig. 1, right). These data argue that CCK-8S and tolbutamide interact in a synergistic fashion. The simplest explanation, based on the available evidence, is that the major effect of CCK-8S is on phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis with the production of inositol 1,4,5-trisphosphate (Ins 1,4,5P3) and diacylglycerol. The major effect of tolbutamide is to stimulate Ca2+ influx, a process which may lead to a further activation of PIP2 hydrolysis [ 17 ]. The CCK-8S-induced rise in Ins 1,4, 5P3 leads to the release of sequestered intracellular [Ca2+]. When this change in intracellular [Ca2+] is coupled to the tolbutamide-induced increase in Ca 2+ influx, the resulting change in Ca2+ metabolism is sufficient to induce the initial phase of insulin secretion. Activation of adenylate cyclase by forskolin (Fig.2, center) causes a considerable enhancement of the insulin secretory response to the combination of CCK-SS plus tolbutamide even though an increase in intracellular [cAMP] causes a partial inhibition of CCK-8S-induced PI hydrolysis [ 18 ]. If the glucose concentration is low (2.75 mmol/1), then CCK-8S alone or tolbutamide alone do not induce a significant increase in insulin secretion [ 11 ], but if combined with forskolin, they will induce a first phase of secretion but no second phase [ 11 ]. Hence, glucose appears to have an independent effect on the metabolism of diacylglycerol. We previously postulated that at a very low glucose concentration (2.75 mmol/1), CCK8S induces an increase in DAG production, but not in DAG accumulation [ 11 ]. Hence, there is no activation of protein kinase C and thus no second phase of insulin secretion. In islets incubated in 5.5 mmol/1 glucose, addition of 10 nmol/1 CCK-8S alone also does not induce any second phase of insulin secretion, but when combined with tolbutamide, it does so. A simple interpretation of this interaction is based on observations in other systems in which it has been shown that both an increase in Ca 2+ influx and an increase in DAG production are necessary to produce a sustained activation of protein kinaseC [ 8 ]. In the present circumstance (5.5 mmol/l glucose), 10 nmol/1 CCK-8S is postulated to cause a small increase in the D A G content of the tissue, but this alone is not sufficient to evoke a significant second phase of insulin secretion. However, when combined with an agent (tolbutamide) that stimulates C a 2+ influx, it does bring about a sufficient activation of C-kinase to induce a second phase of insulin secretion. If under these circumstances MOG, an inhibitor of DAG kinase [15[, is added (Fig.2, left), then an increased accumulation of DAG and a further activation of protein kinase C occur leading to an enhanced secretory response. If the same manipulations are performed on islets incubated in the presence of 1 ~tmol/1 forskolin, then a massive MOG-dependent second phase of insulin secretion is seen (Fig.2, center). All four signals (MOG, CCK-8S, forskolin, and tolbutamide) are necessary for this amplified response: CCK8S induces an increase in both Ins 1,4,5P3 and DAG production, MOG inhibits the further metabolism of DAG, tolbutamide increases Ca 2+ influx, and forskolin raises the cAMP concentration in islets. A remarkable fact is that the only natural secretagogue which appears to produce all four of these biochemical changes is high glucose. It has to be emphasised that the simultaneous presence of moderate glucose level (5.5 mmol/1) controls in an absolute sense the secretory response of the islets to this combination of agonists. If the glucose level is maintained at 2.75 mmol/1, the combination of CCK-SS, forskolin, tolbutamide and MOG induces a weak insulin secretory response. However, at 5.5 mmol/1 glucose, a level of glucose which by itself has rio significant stimulatory effect on insulin secretion, a dramatically amplified, biphasic insulin secretory response occurs in response to MOG, CCK-8S and tolbutamide. An obvious question to be addressed is the nature of the metabolic change induced by glucose which permits such a dramatic difference in responsiveness. In other words, what effector system changes in response to such a small alteration in the ambient glucose level from 2.75 to 5.5 mmol/1, concentrations which by themselves have no significant insulin secretory effect. It is perhaps more than coincidental that almost complete closure of the ATP-sensitive potassium channel occurs between 2.75-6 mmol/1 glucose [ 19 ]. It is tempting to speculate that an increase in ATP levels [ 20 ], brought about by a small! increase in glucose usage rates over this concentration range [ 21 ], promotes closure of these channels and poises the system to respond to a secretory signal. This change, however, is insufficient in and of itself to induce insulin secretion. In other words, the permissive effect of moderate glucose on this agonist combination resides in the ability of glucose, mediated by the generation of ATP, to close ATPregulated potassium channels. Previous work by Dunlop and Larkins [ 22 ] had shown that in neonatal rat islets an increase in glucose concentration leads to the de novo synthesis of diacylglycerol. More recently, Peter-Reisch et al. [ 14 ] have shown that glucose causes a prompt and sustained increase in the tissue content of DAG by a mechanism other than that occurring via PI hydrolysis. Hence, a second important effect of glucose is that of increasing the size of the DAG pool in islets. The fact that MOG causes a further enhancement of glucose-dependent insulin secretion suggests that even at high glucose and hence presumably high rates of dephosphorylation of PA to DAG [23], there are also high rates of DAG kinase activity. Addition of MOG under this circumstance would lead to higher levels of DAG and higher rates of protein kinase C activity. From a practical point of view~ the present results along with our previous study of CCK-8S-glucose interactions provide evidence that it is possible to regulate, by pharmacological means, the separate phases of insulin secretion. In clinical situations characterised by altered temporal patterns of insulin secretion, it might be possible to manipulate B-cell function to normalise insulin secretory patterns. Finally, it must be noted that the foregoing discussion is based on the concept that M O G acts specifically to inhibit the enzyme D A G kinase and, thereby, cause an increase in the D A G content o f B cells. As a result o f this change in D A G content, a further activation o f protein kinase C is thought to occur. These assumptions remain to be validated in this tissue. Acknowledgements. This work was supported by grant AMI9 813 from the National Institutes of Health. The expert secretarial assistance of Ms. N.Canetti is gratefully acknowledged. 1. 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W. S. Zawalich, K. C. Zawalich, H. Rasmussen. The effect of monooleoylglycerol on insulin secretion from isolated perifused rat islets, Diabetologia, 1989, 360-364, DOI: 10.1007/BF00277259