Disappearance of glucose-induced oscillations of cytoplasmic Ca2+ in pancreatic β-cells exposed to streptozotocin or alloxan

Toxicology, 63 (1990) 263--271 Elsevier Scientific Publishers Ireland Ltd.

Disappearance of glucose-induced oscillations of cytoplasmic Ca 2÷ in pancreatic fl-cells exposed to streptozotocin or alloxan Eva Grapengiesser, Erik Gylfe and Bo Hellman Department of Medical Cell Biology, University of Uppsala, Biomedicurn, Box 571, S-751 23 Uppsala (Sweden) (Received March 5th, 1990; accepted May 7th, 1990)

Summary Dual wavelength microfluorometry and the indicator fura-2 were employed for measuring cytoplasmic Ca 2* (Ca2*i) in individual pancreatic fl-cells isolated from ob/ob-mice. In most fl-cells, a rise of external glucose from 3 to 20 mM resulted in large amplitude oscillations in Ca2÷~, superimposed on a basal level of 60--90 nM. The diabetogenic agents streptozotocin and alloxan (1--4.4 mM) rapidly abolished the glucose-induced oscillations of Ca2*~. The presence of a high glucose concentration during the exposure to the drugs counteracted the action of alloxan but not that of streptozotocin. Perturbation of the cyclic variations of Ca2÷~ by streptozotocin did not interfere with a glucose-induced increase of the ion in mildly affected fl-cells. The most advanced lesions obtained with the exposure to the diabetogenic agents were manifested as uncontrolled and sustained increases of Ca2+~. Although disrupting the intracellular Ca 2÷ homeostasis by separate mechanisms, streptozotocin and alloxan may finally kill the fl-cells by activating a common suicidal process due to an excessive rise of Ca2÷~. Key words: Streptozotocin; Alloxan; Cytoplasmic Ca:* oscillations; Pancreatic fl-cells; Glucose

Introduction It has b e e n k n o w n f o r m o r e t h a n a d e c a d e t h a t t h e c i r c u l a t i n g levels o f i n s u l i n are s u b j e c t t o cyclic v a r i a t i o n s , r e f l e c t i n g p u l s a t i l e release o f the h o r m o n e [ 1 - - 3 ] . T h e o s c i l l a t o r y p a t t e r n o f s e r u m i n s u l i n c a n be e x p e c t e d to be o f p h y s i o l o g i c a l s i g n i f i c a n c e in c o u n t e r a c t i n g t h e d o w n r e g u l a t i o n o f t h e insulin r e c e p t o r s [4]. It is " t h e r e f o r e o f i n t e r e s t t h a t a n e a r l y i n d i c a t o r o f d e v e l o p m e n t o f d i a b e t e s is t h e disa p p e a r a n c e o f t h e r e g u l a r cycles o f p l a s m a i n s u l i n , i m p l y i n g t h a t c i r c u l a t i n g insulin will be less e f f i c i e n t [5].

Address all correspondence and reprint requests to: B. Hellman. 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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The cytoplasmic concentration of C a 2÷ (Ca2+i) is the principal intracellular regulator of insulin release from the pancreatic 0-cells [6]. We have recently demonstrated that glucose induces large amplitude oscillations of this calcium [7--10], suggesting that the pulsatility of insulin release is an intrinsic property of the/3cells. To elucidate whether minor damage of the /3-cells may interfere with their inherent pacemaker function, it was studied how the glucose-induced oscillations of Ca2+i are affected by streptozotocin and alloxan. It will be shown that both of these cytotoxic agents effectively remove the cyclic variations of CaZ÷~. In the case of streptozotocin the effect was often associated with a modified response to glucose with elevation of Ca2+~. Materials and methods

Reagents of analytical grade and deionized water were used. Streptozotocin was donated by the Upjohn Company, Kalamazoo, Michigan and alloxan was from Sigma Chemical Company, St. Louis, Missouri. Collagenase, H E P E S and bovine serum albumin (fraction V) were products of Boehringer Mannheim GmbH, Mannheim, F.R.G. Fetal calf serum was provided by Gibco Ltd., Paisley, U.K. and the acetoxymethylester of fura-2 obtained from Molecular Probes Incorp., Eugene, Oregon. Adult, obese-hyperglycemic mice (gene symbol o b / o b ) were taken from a noninbred strain [ll] and starved overnight. Islets of Langerhans were isolated by collagenase from the splenic part of the pancreas and single cells prepared by shaking in a CaE÷-deficient medium [12]. After suspension in RPMI 1640 medium supplemented with 10°70 fetal calf serum, 100 IU/ml penicillin, 100/~g/ml streptomycin and 30/~g/ml gentamycin, the cells were allowed to attach to circular 25mm cover glasses during 1--3 days. Further experimental handling was performed with a basal medium physiologically balanced in cations, with Cl- as the sole anion [13]. When not otherwise stated this medium was supplemented with 3 mM glucose and 0.1 mg/ml albumin. After rinsing, the cells were loaded with fura-2 during 30 min of incubation at 37°C in 0.5 /~M fura-2 acetoxymethylester. The principle of using fura-2 for measuring CaZ+~ in individual cells by dual wavelength fluorometry is based on a shift in the excitation spectrum between free and calcium-complexed indicator [14]. Accordingly, when monitoring the fura-2 fluorescence excited at two wavelengths, alterations of Ca2÷i are manifested as a change in the ratio between the fluorescence at these wavelengths. The ratio technique has the advantage that many fluorescence changes unrelated to Ca2+i are cancelled. The cover glasses with the fura-2 loaded cells were rinsed and used as the bottoms of open chambers designed for microscopy [15]. The chamber wall was a broad silicon rubber ring (9 mm inner diameter) pressed to the cover glass by the threaded chamber mount and a thin stainless steel ring. Cannulas fixed to this ring were connected to a two-channel peristaltic pump allowing steady superfusion of a 2.5-mm medium layer at a rate of 1.0 ml/min. The chamber was placed on the stage of an inverted microscope (Leitz Diavert) within a climate box maintained at 37°C by an air-stream incubator. The microscope was equipped with a

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Ploemopak epifluorescence illuminator and a 100x UV-fluorite objective (Nikon). A 75-W xenon arc lamp combined with 5-nm half-bandwidth interference filters in the rotating air turbine filter changer of a time-sharing multichannel spectrophotofluorometer [16] provided the exciting light flashes of l ms duration at 340 and 380 nm every 10 ms. Emission was measured with a photomultiplier at 510 nm using a 30-nm half-bandwidth filter. The electronically separated signals excited at 340 and 380 nm were fed into an analog ratio meter. The ratio as well as the 380 nm excitation fluorescence were recorded on a strip chart recorder. By determining separately the 340/380 nm fluorescence excitation ratio and the 380 nm excitation fluorescence both in a Ca2÷-deficient medium and at excess concentrations of the ion, it was possible to calculate Ca2÷~ assuming a K d for the Ca 2÷fura-2 complex of 224 nM [14]. Previous studies have indicated that the islets employed contain more than 90o7o /3cells [11], which respond normally to glucose and other stimulators of insulin release [17]. Selection of/l-cells for analyses were based on described criteria [9,18], initiating the experiments with the generation of glucose-induced oscillations of Ca2÷i. After a period of superfusion the /3-cells were exposed to streptozotocin or alloxan during the rest of the experiments. The indicated concentrations of the compounds refer to those initially added, neglecting their rapid inactivation at a physiological pH [19]. Results

In a great majority of the /3-cells raising the glucose concentration from 3 to 20 mM resulted in large amplitude oscillations of Ca2*i superimposed on a basal level of 60--90 nM. The periodicity of the oscillations varied between 2 and 6 min. The glucose-induced oscillations of Ca2*~ were abolished by streptozotocin. When added at a concentration of 2.2 mM, this nitrosourea compound caused the disappearance of the oscillations in all experiments (n = 17). Three types of reactions were recognized with regard to the concomitant modifications of the Ca2*~ levels. (A) In the majority of the experiments (n = 11) the disappearance of the oscillations was associated with a sustained increase of Ca2*i at a level below the peak of the oscillations (Fig. 1). This elevation of Ca2*~ was glucose-dependent, as indicated from the return to basal or close to basal levels when lowering the concentration of the sugar to 3 mM. (B) In some experiments (n = 4) the disappearance of the oscillations was associated with lowering to basal or slightly elevated Ca2*i, which remained unaffected when decreasing the sugar concentration (Fig. 2). (C) Occasionally (n = 2) streptozotocin induced a large rise of Ca2*~ independently of the glucose concentration (not shown). When testing concentrations of streptozotocin between 0.5 and 4.4 mM it became evident that the above-mentioned types of reactions represented increasing damage to the /3-cells. Whereas stage A was typical for the exposure to low concentrations of streptozotocin, stage C reflected the pronounced lesions obtained when the /3-cells were exposed to higher concentrations of the diabetogenic agent (Fig. 3). The effect of tolbutamide was tested in 8 experiments where streptozotocin had abolished most of the glucose-induced rise of Ca2*~ (Fig. 4).

265

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Fig. 1. Suppression of glucose-induced oscillations of cytoplasmic Ca 2÷ by streptozotocin. The arrow indicates the time of addition of 2.2 mM streptozotocin and the bar the period during which glucose was raised from 3 to 20 mM. The exposure to streptozotocin caused the disappearance of the oscillations with maintenance of Ca2÷, at an elevated level which was reversed by lowering the glucose concentration.

W h e r e a s t h e r e was a n i m m e d i a t e rise o f Ca2+~ in 5 cells e x p o s e d to 1 0 0 / a M o f this s u l f o n y l u r e a ( p a n e l A ) , t h e r e m a i n i n g o n e s d i d n o t r e s p o n d ( p a n e l B). I n e x p e r i m e n t s p e r f o r m e d like t h o s e w i t h s t r e p t o z o t o c i n , 2 m M a l l o x a n f a i l e d to a f f e c t t h e o s c i l l a t i o n s o f Ca2÷i i n d u c e d by 20 m M g l u c o s e (Fig. 5). H o w e v e r , e x p o s u r e o f g l u c o s e - r e s p o n s i v e f3-cells t o t h e s a m e c o n c e n t r a t i o n o f a l l o x a n in t h e p r e s e n c e o f 3 m M o f t h e s u g a r (Fig. 6) r e s u l t e d in a p a r t i a l ( p a n e l A ) o r c o m p l e t e ( p a n e l B) s u p p r e s s i o n o f t h e o s c i l l a t i o n s n o r m a l l y o b t a i n e d w h e n r a i s i n g the gluc o s e c o n c e n t r a t i o n to 20 m M . In a d d i t i o n to i n t e r f e r i n g w i t h the a b i l i t y o f glu-

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of glucose-induced oscillations of cytoplasmic Ca z÷ after adding alloxan to a 20 mM glucose. The arrow indicates the time of addition of 2 mM alloxan and during which glucose was raised from 3 to 20 raM. Representative for results experiments.

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Fig. 6. S u p p r e s s i o n of g l u c o s e - i n d u c e d o s c i l l a t i o n s o f Ca2+~ after a d d i n g a l l o x a n to a m e d i u m containing 3 m M glucose. T h e a r r o w s i n d i c a t e the t i m e o f a d d i t i o n s of 2 m M a l l o x a n a n d the bars the periods d u r i n g which glucose was raised f r o m 3 to 20 m M . The e x p o s u r e to a l l o x a n c a u s e d a progressive rise o f Ca2÷, a s s o c i a t e d w i t h p a r t i a l (panel A ; n = 6) or c o m p l e t e (panel B; n = 2) s u p p r e s s i o n of the g l u c o s e - i n d u c e d o s c i l l a t i o n s o f the ion.

268

cose to induce high amplitude oscillations, the addition of 2 mM alloxan to a medium containing 3 mM glucose resulted in a progressive increase of Ca2+i . The latter effect became more pronounced at higher concentrations of the drug (not shown). Discusssion

Streptozotocin and alloxan are widely used for inducing experimental diabetes based on selective destruction of the pancreatic g-cells [19--21]. It is well established from various experiments that both agents rapidly damage the B-cells. The mechanisms involved in their cytotoxic action still remain to be fully elucidated. A common mechanism has been proposed involving lowering of the NAD levels [22] following breaks of DNA strands [23]. However, NAD-requiring DNA repair synthesis was found after exposure to streptozotocin but not to alloxan [24]. Moreover, only streptozotocin induced prolonged suppression of the secretory activity in surviving g-cells from islets kept in culture [25,26]. As indicated from the present study both alloxan and streptozotocin rapidly suppressed the glucose-induced oscillations of Ca2+i . In the case of alloxan this effect occurred when the g-cells were exposed to the agent in the presence of a low but not high glucose concentration. This observation reinforces previous arguments for a protective effect of the sugar against the toxic action of alloxan [21,27,28]. The depolarization mediating glucose stimulation of insulin release can be attributed to closure of a specific type of K ÷ channel by A T P [6,29]. Since streptozotocin has been reported to interfere with glucose metabolism in the pancreatic B-cells [25], lower their content of A T P [30] and suppress the electrical activity [31], it is not surprising that the exposure to this nitrosourea has been found to result in a defective glucose response with an insulin release pattern similar to that seen during the development o f insulin-dependent diabetes [25]. Sulfonylureas mimic the action of A T P in closing the K* channels involved in glucose depolarization of the g-cells [32,33]. Accordingly, tolbutamide often induced an immediate rise of Ca2÷i in g-cells which had lost their responsiveness to glucose after streptozotocin treatment. It was evident from the present study that experimentally induced lesions of the g-cells can abolish glucose-induced oscillations of Ca2÷i with maintenance of a sugar response manifested as a raised concentration of the ion. Accordingly, concentrations of streptozotocin slightly exceeding those required for breaking the cyclic variations, stabilized Ca2÷i at an elevated level as long as the g-cells were exposed to raised concentrations of glucose. In the absence of detailed knowledge about the control mechanisms for the pulsatile insulin release, it is difficult to evaluate how the loss o f the pacemaker function of the g-cells contributes to the development of diabetes. However, it is pertinent to note that disappearance of the periodic variations of circulating insulin has been considered to be an early indicator of diabetes in man [5]. The most advanced damage seen when exposing the g-cells to streptozotocin and alloxan was an excessive rise of Ca2÷i unaffected by the glucose concentration. It is well established that uncontrolled and sustained increases of Ca2÷i can

269

i n i t i a t e c y t o t o x i c p r o c e s s e s . S t u d i e s o f o t h e r k i n d s o f cells h a v e i n d i c a t e d C a 2÷induced suicidal processes including endonuclease activation with extensive DNA f r a g m e n t a t i o n [34]. T h e c o n c e p t t h a t t h e 3-cell d e s t r u c t i o n e v o k e d b y a l l o x a n a n d s t r e p t o z o t o c i n m a y u l t i m a t e l y be d e p e n d e n t o n a n e x c e s s i v e rise o f Ca2+i is n o t at v a r i a n c e w i t h t h e n o t i o n t h a t t h e i r c y t o t o x i c i t y is i n i t i a l l y m e d i a t e d by d i f f e r e n t mechanisms.

Acknowledgements The authors are indebted to Ms Hel6ne Dansk for expert technical assistance a n d to D r s S t e l l a n S a n d i e r , D e c i o E i z i r i k a n d E v a S t r a n d e l l f o r h e l p f u l a d v i c e . T h e s t u d y was s u p p o r t e d by g r a n t s f r o m t h e S w e d i s h M e d i c a l R e s e a r c h C o u n c i l (12x-562; 12x-6240), t h e S w e d i s h D i a b e t e s A s s o c i a t i o n , t h e N o r d i c I n s u l i n F o u n dation, the Family Ernfors Foundation and the Swedish Hoechst and Novo C o m p a n i e s . S t r e p t o z o t o c i n was a g i f t f r o m D r D o u g l a s R. M o r t o n , J r . , U p j o h n Co., Kalamazoo, Michigan.

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