Ailoxan-Induced Alteration of Insulin Release, Rubidium Efflux and Glucose Metabolism in Rat Islets Stimulated by Various Secretagogues

Transcription

1 Dabetologa 16, (1979) Dabetologa 9 by Sprnger-Verlag 1979 Aloxan-Induced Alteraton of Insuln Release, Rubdum fflux and Glucose Metabolsm n Rat Islets Stmulated by Varous Secretagogues J. C. Henqun, P. Malvaux, and A.. Lambert Unt6 de Dabbte et Crossance, Unversty of Louvan School of Medcne, UCL 54.74, Brussels, Belgum Summary. Insuln release and 86Rb efflux were studed n perfused rat slets exposed n vtro to alloxan (2 mmol/1) for 5 mn. At a low glucose concentraton, alloxan transently ncreased 86Rb efflux. Alloxan mmedately and completely abolshed the secretory response to glucose (15 mmol/1) and markedly delayed the reducton n 86Rb efflux normally produced by the sugar. 3-0-methylglucose (20 mmol/ 1) provded complete protecton aganst the alteraton of 86Rb efflux and partal protecton aganst the nhbton of nsuln release. Immedately after alloxan treatment, glyceraldehyde, ct-ketosocaproc acd and tolbutamde stll nduced a rapd release of nsuln, but the late phase normally stmulated by glyceraldehyde and a-ketosocaproc acd was nhbted. If slets were exposed to glyceraldehyde or tolbutamde 15 mn after alloxan treatment, the rapd nsuln release was also markedly mpared. Alloxan faled, however, to affect the ablty of these three stmul to reduce 86Rb efflux from slet cells. Glucose oxdaton and utlzaton were decreased n alloxantreated slets and 3-0-methylglucose protected aganst ths effect. The results show that the glucose recognton system n B-cells s the most rapdly and severely affected by alloxan. The drug also alters the response to other secretagogues, the nsuln releasng propertes of whch can be mpared wthout alteraton of ther ablty to reduce 86Rb efflux. Key words: Isolated rat slets, alloxan, perfuson, nsuln release, rubdum efflux, glucose metabolsm, glucose, glyceraldehyde, a-ketosocaproc acd, tolbutamde. Alloxan has been wdely used to nduce expermental dabetes [1], but the exact mechansms by whch the drug produces a relatvely selectve destructon of pancreatc B-cells reman elusve. lucdaton of the molecular mechansms of ts acton would certanly be of more than pharmacologcal nterest. Indeed, observatons that glucose can protect B-cells aganst alloxan toxcty [2, 3] make the drug a potental probe of the physologcal stmulus-secreton couplng. Present expermental evdence, recently dscussed n detal [4], suggests that the B-cell membrane may be an mportant ste of the acton of alloxan. In mouse slets alloxan produces depolarzaton of B- cells [5] and nhbton of the unvalent caton pump [6]. In rat slets t produces a loss of membraneassocated partcles [7]. In toadfsh slets [8], but not n rat slets [9], t ncreases the membrane permeablty to manntol, whch s normally restrcted to the extracellular space. Recent studes have shown that glucose rapdly and markedly decreases potassum efflux from rat slet cells [10]. Ths supports the suggeston [11] that the depolarzaton of B-cells produced by the sugar [12-13] mght be due to a reducton n K-permeablty. The mportance of these changes n K-permeablty s underlned by the evdence that pharmacologcal agents whch potentate the effect of glucose on K-permeablty also potentate ts nsuln releasng effect, whereas those whch augment K-permeablty antagonze the nsulnotropc acton of the sugar [14-16]. In the present experments, perfused solated rat slets were exposed to alloxan for 5 mn [17] n order to (a) compare the nhbtory effects of the drug on nsuln release stmulated by varous agents, (b) determne whether any alteraton of the effect of these secretagogues s related to an mparment of ther property to change the K-permeablty of slet cells (evaluated by changes n 86Rb efflux), and (c) nvestgate the effects of alloxan on glucose metabolsm by slet cells X/79/0016/0253/$01.60

2 , 254 J. C. Henqun et al.: Alloxan Alteraton of B Cell Propertes 0.03 g, *~ 0.02 ~yo. Alloxon 2ram t-... ~ ~ --o-... ] -o~, " l' ~~,,,~ 510 I K TIM (ran) Fg. 1. ffect of alloxan on S6Rb efflux from rat slets perfused n the presence of 3 mmol/l glucose throughout. The stppled lne shows the rate of a6rb efflux from control slets not treated wth alloxan. As ndcated by the arrows, alloxan (2 mmol/1) was added for 5 mn (40-45 mn 0--0) or 15 mn (40-55 o---o). In the lastmentoned seres of experments, a new medum wth freshly dssolved alloxan was used every 5 mn. Values are means _+ SM of 4 experments Materals and Methods Anmals and Solutons All experments were performed wth slets solated from male Wstar rats ( g) kept on a standard pellet det and tap water ad lbtum. They were klled 3 h after ntrapertoneal njecton of plocarpne (20 mg/kg). The slets were obtaned by combnng mld collagenase dgeston of the gland (5 mg collagenase per pancreas and shakng for 3.5 to 4 mn) and mcrodssecton of the partally dgested peces. Control experments have shown that pretreatment of the anmals wth ths dose of plocarpne dd not sgnfcantly alter the nsuln response of the solated slets to varous secretagogues. The medum utlzed was Krebs-Rnger bcarbonate buffer [18], ph 7.4, gassed wth O2/CO2 (94:6), supplemented wth 0.5% (w/v) bovne serum albumn and contanng 3 mmol/1 glucose n basal condtons. For metabolc studes, 10 mmol/1hps (N-2- hydroxyethylpperazne-n'-2-ethanesulfonc acd) was added to the medum to prevent any change of ph n the small volumes of solutons used n these experments. Because of rapd nactvaton at 37 ~ alloxan (2 mmol/1) was dssolved n the approprate prewarmed medum mmedately pror to use. Ths concentraton of alloxan decreased the ph of the buffer by less than 0.2 unts and a 5 mn treatment wth a medum acdfed to 7.2 (by addton of HC1) dd not affect the slet responses. Control experments were also carred out wth nactvated alloxan (3 h n soluton at 37 ~ before use). Measurements of Insuln Release, Rubdum fflux and Glucose Metabolsm The perfuson technque utlzed to study nsuln release and the method for measurement of mmunoreaetve nsuln (IRI) have been prevously reported [18]. The technque to montor the efflux of S6Rb from preloaded slets was smlar to that recently descrbed [10, 14]. Brefly, groups of 60 to 90 slets were frst loaded wth 86Rb by ncubaton for 150 ran n 0.5 ml basal medum supplemented wth 0,2 mmol/l 86RbC1. They were then washed three tmes wth 2 ml nonradoactve medum and placed n the perfuson chambers (0.3 ml), to whch the perfusate, at 37 ~ was conveyed at a rate of 1.1 ml/mn, a6rb n the effluent fractons (collected at 1 or 2 mn ntervals; 1500 to cpm per fracton) and remanng n the slets at the end of the experment was counted by the Cerenkov radaton [10, 16]. For each collecton nterval, the fractonal efflux of S6Rb (S6Rb released durng tme nterval/86rb remanng n tssue durng that tme nterval) was calculated. Glucose utlzaton by slet cells was measured by the producton of [3H]-water from D-[5-3H] glucose [19] and glucose oxdaton by the converson of D-[U-14C] glucose to [14C]-COz. Pools of slets were frst prencubated at 37 ~ n 10 ml medum contanng glucose alone (3 mmol/1) or wth 3-0-methylglucose (20 mmol/1). After 25 ran, 5 ml of the same prewarmed medum wth or wthout 6 mmol/1 freshly dssolved alloxan was added; n the test groups the fnal concentraton of alloxan was thus 2 mmol/l. Fve mn later, the slets were rapdly and extensvely washed at room temperature before beng transferred to the ncubaton tubes for 1 h at 37 ~ n the followng condtons: (a) utlzaton studes: 10 slets n 20 gl of medum contanng 3 or 15 mmol/1 D-[5-3H]- glucose (2.5 and 0.5 mc/mmol, respectvely), b) oxdaton studes: 10 slets n 50 ~tl of medum contanng 3 or 15 mmol/1d-[u-~4c] - glucose (6 and 1.2 mc/mmol, respectvely). Other techncal aspects of these methods have been descrbed elsewhere [18]. Chemcals Alloxan monohydrate, 3-0-methyl-D-glucose and a-ketosocaproc acd were obtaned from Sgma Chemcal Co., St. Lous, Mo., USA; HPS was from Brtsh Drug Houses, Poole, Dorset, U. K.; D-glyceraldehyde was from Koch-Lght Laboratores Ltd., Colnbrook Bucks, U. K.; collagenase (type IV; 200 U/mg) was from Worthngton Bochemcal Co., Freehold, N. J., USA; tolbutamde was suppled by Hoechst Research Laboratores, Frankfurt/Man, Germany. All other reagents were of analytcal grade and purchased from Merck A. G., Darmstadt, Germany. The radoactve sotopes S6RbC1 ( mc/mmol), D-[U-14C] glucose (327 mc/mmol) and D-[5-3H] glucose (1 C/mmol) were obtaned from the Radochemcal Centre, Amersham, U. K. Presentaton of Results All results are expressed as means -+ SM. The statstcal sgnfcance of dfferences between expermental groups was assessed by Student's t test for unpared data and the Wlcoxon rank-sum test. Results ffect of Alloxan at a Low Glucose Concentraton In control slets perfused wth a medum contanng 3 mmol/1 glucose, the rate of 86Rb efflux slowly decreased wth tme (Fg. 1). xposure of the slets to 2 mmol/1 alloxan for 5 ran, at the same glucose concentraton, had no sgnfcant effect on nsuln release (not llustrated), but ncreased the rate of 86Rb efflux. Ths ncrease n efflux rate was transent, even when

3 J. C. Henqun et al.: Alloxan Alteraton of B Cell Propertes 255 c 0.03 G3 mm ~ 1 4 G 15 mm c 003 G 3ram c" G3mM ~ 3~0_MG_ 20ram F'-1: I..., w ".. G 15 mm g~ "6 0.0;... -~,~... k,<. 2 "6 0.02,t- "~~~ :,-b-~4; I3,t I r 0.01 I I 9. 0.t == 0. --O ~ * ~ - -~,'._~ 0./. ~ 0.2 0, l.o TIM {mn) Fg. 2. ffect of alloxan on the reducton of 86Rb efflux (upper panel) and the stmulaton of nsuln (IRI) release (lower panel) by glucose. Glucose concentraton was ncreased from 3 to 15 mmol/1 from 40 to 80 mn. As ndcated by the vertcal arrows, alloxan (2 mmol/1) was added for 5 mn (35-40 o---o). In ths and all the followng fgures, the stppled lne n the upper panel shows the rate of 86Rb efflux from slets treated wth alloxan between mn 35 and 40 and mantaned n 3 mmol/1 glucose durng the whole experment. Values are means + SM of 4 experments for 86Rb efflux and 5 experments for nsuln release / TIM (mn) Fg O-Methylglucose (3-0-MG) protecton aganst alloxan alteraton of the reducton of 86Rb efflux (upper panel) and the stmulaton of nsuln (IRI) release (lower panel) by glucose. Glucose concentraton was ncreased from 3 to 15 mmol/1 from 40 to 80 mn. 3-0-MG (20 retool/l) was present 5 mn before and durng alloxan treatment. As ndcated by the vertcal arrows, alloxan (2 mmol/1) was added for 5 mn (35-40). The broken lnes n upper and lower panels show the control effect of 15 retool/1 glucose, wthout pror alloxan treatment. The stppled lne n the upper panel as n Fg. 2. Values are means _+ SM of 4 experments for 86Rb efflux and 5 experments for nsuln release alloxan remaned present for 15 mn (a freshly prepared soluton was used every 5 mn). ffect of Alloxan on Glucose Stmulaton As shown n Fg. 2, stmulaton wth 15 mmol/1 glucose produced a marked fall n the rate of 86Rb efflux from control slets (upper panel) and a concomtant bphasc ncrease n nsuln release (lower panel). Alloxan treatment for the last 5 mn precedng glucose stmulaton abolshed nsuln secreton (lower panel) and prevented glucose from rapdly decreasng the K-permeablty n the slet cells. Thus, durng the frst 15 mn of stmulaton wth 15 mmol/1 glucose, the rate of 86Rb efflux remaned the same as n the presence of 3 mmol/1 glucose; however, t fnally reached the same low level as n control slets after 30 mn of stmulaton (Fg. 2, upper panel). Treatment of the slets wth nactvated alloxan n the presence of 3 mmol/1 glucose had no effect on 86Rb efflux and dd not alter the effect of a hgh glucose concentraton (not shown). xposure of the slets smultaneously to 2 mmol/1 alloxan (for 5mn) and 15 mmol/1 glucose only resulted n a partal decrease n the nsuln-releasng effect of the sugar. The frst and second phases of nsuln release were nhbted by 59 and 51% (P < 0.005, n --- 5), respectvely. In these condtons, 86Rb efflux rate was not ncreased by alloxan but was reduced by hgh glucose, as n control slets (the fracton of 86Rb lost per mnute was _ after 5 mn of 15 mmol/1 glucose and 2 mmol/l alloxan together, as compared to _ after 5 mn of 15 retool/1 glucose alone; n = 3). In the experments llustrated n Fg. 3, 3-0- methylglucose (20 mmol/1) was added to the perfusate 5 mn before alloxan and was mantaned durng the 5 mn of exposure to the drug. The presence of 3-0-methylglucose dd not change basal nsuln release, but provded partal protecton aganst nh-

4 256 J. C. Henqun et al.: AUoxan Alteraton of B Cell Propertes o g o 14. G 3raM... f I... +,.,. /... +, r : "?4... L_ l, ' G 3mM + Glyceraldehyde lomm!t, ""'.+.!~ "-.. ~ + 'r o. <_.o. % - *'"P-,--A-.,"~-6--o g, = ut 0.01 &- -o..~,. G3 mm ~+ t ;+1,s!":... :l,s,,,! G3mM " % "...+. *KIC 10ram I I I 0.01 I I A o '1?/'~, ~..+_.o.-~--.~... ',+,~ ~,.~.f -,"....., ' Id ~,.+._,.C... ~ L TIM In,n) Fg. 4. ffect of alloxan on the reducton of 86Rb efflux (upper panel) and the stmulaton of nsuln (IRI) release (lower panel) by D-glyceraldehyde. Glucose concentraton was 3 mmol/1 throughout and D-glyceraldehyde (10 mmol/1) was present from 40 to 80 ran. As ndcated by the vertcal arrows, alloxan (2 mmol/1) was added for 5 mn ( (35-40 o---o). The stppled lne n the upper panel as n Fg. 2. Values are means +_ SM of 5 control and 3 alloxan experments for S6Rb efflux and of 8 control and 5 alloxan experments for nsuln release ~ :... : : TIM Imn) Fg, 5. ffect of alloxan on the reducton of 86Rb efflux (upper panel) and the stmulaton of nsuln (IRI) release (lower panel) by a-ketosocaproc acd (KIC). Glucose concentraton was 3 mmol/l throughout and a-ketosocaproc acd (10 mmol/1) was present from 40 to 80 mn. As ndcated by the vertcal arrows, alloxan (2 mmol/l) was added for 5 mn ( o---o). The stppled lne n the upper panel as n Fg. 2. Values are means _+ SM of 3 experments for S6Rb efflux and 4 experments for nsuln release bton of the glucose effect by alloxan (Fg. 3, lower panel). The frst and second phases of nsuln secreton were nhbted by 57 and 32% respectvely (P < 0.01 and P < 0.02, n = 5). 3-0-Methylglucose tself dd not alter the rate of 86Rb efflux from the slets, but prevented the ncreased efflux otherwse produced by alloxan. It also protected the ablty of a hgh glucose concentraton to reduce 86Rb efflux rapdly (Fg. 3, upper panel). However, the changes n 86Rb efflux and nsuln release produced by 15 mmol/1 glucose after exposure to alloxan together wth 3-0-methylglucose were slghtly delayed as compared to controls. ffect of Alloxan on D-Glyceraldehyde Stmulaton In control slets, 10 mmol/1 glyceraldehyde produced a rapd decrease n 86Rb efflux and a smultaneous bphasc release of nsuln (Fg. 4). Alloxan treatment durng the last 5 mn precedng stmulaton delayed the early phase of release nduced by the trose and markedly nhbted the late phase. If smlar exposure to auoxan occurred earler n the washng perod and was followed by 15 mn n basal medum, glyceraldehyde stmulaton was almost neffectve (Fg. 4, lower panel, and Table 1). On the other hand, Fg. 4 (upper panel) shows that the rate of 86Rb efflux, ncreased by alloxan, was quckly reduced upon addton of glyceraldehyde. Ths effect, although somewhat delayed as compared to controls, occurred much earler than after stmulaton wth 15 retool/1 glucose (compare wth Fg. 2) and was present even when the secretory response was almost suppressed. The presence of 3-0-methylglucose durng alloxan treatment completely protected the slets aganst nhbton of the nsulnotropc acton of glyceraldehyde (Table 1). The two phases of nsuln release stmulated by glyceraldehyde were reduced by about 40% when no glucose was present n the medum and

5 J. C. Henqun et al.: Alloxan Alteraton of B Cell Propertes 257 were almost completely abolshed f alloxan exposure occurred n these condtons (Table 1). ffect of Alloxan on a-ketosocaproc Acd Stmulaton Stmulaton wth a-ketosocaproc acd (10 mmol/1) resulted n a concomtant decrease n the rate of 86Rb efflux and a bphasc ncrease n nsuln secreton (Fg. 5). Immedately after exposure of the slets to alloxan, a-ketosocaproc acd stll nduced a slghtly smaller rapd phase of nsuln release ( ng/slet vs ng/slet n controls, P < 0.05, n = 4) followed by a markedly depressed late phase (lower panel) and remaned able to reduce rapdly the rate of 86Rb efflux (upper panel) ] 0.03 g "s 0.02 o I... 1 v+,, G3 mm ~_o-] ~, ol 9,," *, g "~,... ~ 9 '.w. ~,... '.~ "... G3mM Tolbutamde 200pg/ml ~ %",. I I 1 ffect of Alloxan on Tolbutamde Stmulaton At the glucose concentraton used (3 mmol/1), tolbutamde (200 gg/ml) stmulated a monophasc release of nsuln and decreased the rate of S6Rb efflux (Fg. 6). Alloxan treatment of the slets mmedately before stmulaton dd not sgnfcantly affect the releasng acton of the sulphonylurea (1.04 +_ 0.08 ng/slet, n = 5 vs ng/slet n controls, n = 8). By contrast, the nsuln response to tolbutamde was markedly reduced (0.40 _ ng/ slet, n = 5, P < 0.001) f smlar exposure to alloxan had occurred earler n the washng perod (lower panel). In addton, n a glucose-free medum, a 5 mn treatment of the slets wth alloxan nhbted the releasng effect of tolbutamde when tested 15 mn later (0.17 _ ng/slet vs0.37 _ ng/ slet n controls, P < 0.02, n = 5). As shown n the upper panel of Fg. 6, the rapd reducton n 86Rb ~_ 02 I... ; f-o-] _~. ~... ~ "A" ""':.~:&~~_+~ ""~'-'-.'29, -.. -,~-.--A4 I I I TIM (rnn) Fg. 6. ffect of alloxan on the reducton of S6Rb efflux (upper panel) and the stmulaton of nsuln (IRI) release (lower panel) by tolbutamde. Glucose concentraton was 3 mmol/1 throughout and tolbutamde (200 gg/ml) was present from 40 to 80 mn. As ndcated by the vertcal arrows, alloxan (2 mmol/l) was added for 5 mn ( (35-40 o---o). The stppled lne n the upper panel as n Fg. 2. Values are means SM of 5 control and 3 alloxan experments for 86Rb efflux and of 8 control and 5 alloxan experments for nsuln release Table 1. ffect of alloxan on the stmulaton of nsuln release by D-glyceraldehyde xp n xpermental condtons (mmol/1) IRI release durng stmulaton (ng/slet) Glucose Alloxan 3-0-methylglucose mn ran _+0.00 a a b a a,c 1.36 a,c d d Immedately after solaton groups of 30 slets were transferred nto the per chambers for 80 mn. Glyceraldehyde (10 mmol/1) was added to the medum from ran 40 to 80. The glucose concentraton ndcated n the table remaned the same throughout. Alloxan treatment lasted 5 mn (mn 20-25) In expermental seres 5, 3-0-methylglucose was present 5 ran before and after as well as durng alloxan treatment, thus from mn 15 to 30 Values are means _+ SM of n experments. Sgnfcance levels: a p < vs xp 1; b p < 0.01 vsxp 1; c p < vsxp 3; d NS, P > 0.05 vs xp 3

6 258 J. C. Henqun et al.: Alloxan Alteraton of B Cell Propertes Table 2. ffect of alloxan on glucose metabolsm by solated slets xpermental Glucose utlzed Glucose oxdzed condtons durng (pmol/h/slet) (pmol/h/slet) prencubaton (mmol/1) Glucose 3 mmol/l Controls Alloxan (2) 35.0_+0.9 (8) a (8) 11.1_+0.9 (8) 5.6_+0.7 a (8) Glucose 15 rnrnol/l Controls (11) 44.8_+2.5 (11) Alloxan (2) a (11) a (11) Alloxan (2) _+3.2 b (9) (9) 3-0-methylglucose (20) Values are means _+ SM of the number of batches of slets gven n parentheses. Sgnfcance levels: a p < vs approprate controls at the same glucose concentraton; b p < vscontrols and alloxan alone, n the presence of 15 mmol/1 glucose efflux by tolbutamde perssted after alloxan treatment, even when secreton of nsuln was nhbted. ffect of Alloxan on Glucose Metabolsm In vtro treatment of the slets for 5 mn wth 2 mmol/1 alloxan n the presence of 3 mmol/1 glucose reduced glucose utlzaton and oxdaton by roughly 50% both at a low (3 mmol/1) and a hgh (15 mmol/1) glucose concentraton (Table 2). The presence of 3-0-methylglucose (20 retool/l) durng exposure to alloxan afforded partal protecton of glucose utlzaton and complete protecton of glucose oxdaton. Dscusson ffect of Alloxan on Insuln Release In contrast to the well documented alloxan nhbton of glucose-stmulated nsuln release, the effects of the drug on the response to other secretagogues have not been extensvely studed. It has been reported that, after alloxan treatment n vtro, the rapd release of nsuln stmulated by tolbutamde s preserved [17], whereas the response to leucne s partally nhbted [20]. The effects of alloxan on the stmulaton by glyceraldehyde do not seem to have been nvestgated. It s known, however, that nether glyceraldehyde nor tolbutamde protects aganst the n vvo dabetogenc acton of alloxan [21] nor aganst the n vtro alteraton of glucose-stmulated nsuln release [22] and pronsuln bosynthess [23]. The present experments demonstrate that the secretory response of B-cells to dfferent stmul s not unformly mpared mmedately after alloxan. As prevously reported [17] treatment of the slets wth the drug durng the 5 ran mmedately precedng stmulaton s suffcent to abolsh the response to glucose. By contrast, tolbutamde, glyceraldehyde and a-ketosocaproc acd reman able to nduce an almost normal rapd release after such treatment, the second phase of secreton stmulated by the two latter secretagogues beng clearly reduced. Yet the dfferental effect of alloxan on the rapd and late phases of nsuln release s only apparent. Thus, the early responses to tolbutamde and glyceraldehyde were also markedly nhbted f the stmulaton was appled 15 mn after alloxan treatment. Snce these experments were carred out n the presence of 3 mmol/1 glucose, the possblty was consdered that the effect of the secretagogue tself was not markedly affected by alloxan, but that only the potentaton by the low glucose concentraton was suppressed. Such a vew s unlkely to explan the effects on a-ketosocaproc acd stmulaton, snce the releasng propertes of ths leucne dervatve are not augmented by a low glucose concentraton [24]. Furthermore, t cannot account for the effect of alloxan on glyceraldehyde or tolbutamde stmulaton; thus, the release of nsuln nduced by the trose n complete absence of glucose s also nhbted by alloxan and s much hgher than that occurrng after alloxan treatment n 3 mmol/1 glucose (Table 2). Smlarly, the nsuln response to tolbutamde n a glucose-free medum s decreased by alloxan. It appears that glucose stmulaton of nsuln release s by far the most senstve to alloxan damage, but that the drug also produces a delayed nhbton of release when stmulated by agents structurally unrelated to glucose. ffect of Alloxan on Rubdum fflux Because of ts longer half-lfe, 86Rb s a convenent substtute for 42K, partcularly when qualtatve changes n the K-permeablty are studed [10, 14, 16]. At a low glucose concentraton, nether 42K [10] nor 86Rb efflux from the slet cells follows smple exponental knetcs (whch would gve a horzontal curve wth the present mode of expresson). The present experments confrm that glucose [10, 11, 14] and tolbutamde [25] markedly reduce the potassum permeablty n slet cells. They further demonstrate that the same effect s produced by glyceraldehyde and a-ketosocaproc acd. Ths suggests that the four agents (provded a-ketosocaproc acd has the same electrcal propertes as leucne) depolarze B cells [26, 27] by a smlar mechansm. By contrast, exposure of the slets to alloxan produced an ncrease n the rate of 86Rb efflux prevented by a hgh concentraton of 3-0-methylglucose or glu-

7 J. C. Henqun et al.: Alloxan Alteraton of B Cell Propertes 259 cose. Such an effect of auoxan was not found n slets of ob/ob mce [6], probably because measurements of the amount of 86Rb retaned n the tssue are not senstve enough to detect a transent ncrease n 86Rb efflux. Although ths ncrease n K-permeablty could concevably be the result of damage to the cell membrane, several arguments do not support ths possblty: (a) the effect s transent even when alloxan s present for 15 mn; (b) t s not accompaned by a leak of nsuln n our system; (c) a smlar dose of auoxan does not augment the permeablty of rat slet cell membranes to sucrose [9]; and (d) t s quckly reversed by glyceraldehyde, tolbutamde and a- ketosocaproc acd. We would suggest rather that the blockade [6] of an electrogenc Na/K pump [27] s, at least partally, the cause of the depolarzaton measured n alloxan treated B-cells [5] and that the ncrease n 86Rb efflux s the consequence of ths fall n membrane potental. The possble nterrelatonshps between onc, electrcal and metabolc changes produced by alloxan are dffcult to evaluate. For nstance, an arrest of the Na/K pump can theoretcally be the cause as well as the consequence of decreased metabolsm. However, the depolarzaton of B-cells produced by alloxan s not expected to result from an mpared energy producton, snce classcal uncouplers or nhbtors of glucose metabolsm hyperpolarze B-cells [12, 28]. A drect effect of alloxan on the Na/K pump s thus plausble [6]. Smlar to ts nsuln releasng propertes, the ablty of glucose to reduce K-permeablty n slet cells appears exqustely senstve to alloxan. Thus, among the four agents tested, only glucose was unable to reduce rapdly 86Rb efflux from slets ntally exposed to alloxan. Snce normal glucose metabolsm s requred for glucose to decrease K-permeablty [10], alloxan-nduced metabolc alteratons mght be nvolved. In several expermental condtons alloxan barely affected the ablty of secretagogues to reduce K-permeablty n slet cells, whereas t markedly altered ther nsuln releasng effect. Such a dssocaton has also been found n the absence of extracellular calcum [10] and suggests that the alloxan block of nsuln release s not at the earlest steps of stmulussecreton couplng. However, a complete understandng of the mechansms underlyng secretagoguenduced changes n K-permeablty s obvously needed to propose a ratonal explanaton of the present observatons. ffect of Alloxan on Glucose Metabolsm The present results clearly show that a bref alloxan treatment n vtro severely alters both glucose utlzaton ("glycolyss") and oxdaton n slet cells Methylglucose protects aganst ths deleterous effect, whch cannot be accounted for by a reduced glucose transport n slet cells [9]. An mparment of glucose oxdaton by mouse slets has also been reported after n vvo treatment of the anmals wth alloxan [29] or n vtro exposure of the slets to the drug [30]. It s clear, however, that detaled tme course studes are necessary to establsh whether there s a causal relatonshp between the alteraton of glucose metabolsm and the nhbton of nsuln release. Nevertheless, these observatons and the evdence of a strong nhbton of pronsuln bosynthess n solated slets after n vvo [31] or n vtro [23, 32] treatment by alloxan show that, besdes ts membrane effects, atloxan also has ntracellular effects. Mechansm of Alloxan Acton Although t remans dffcult to propose a complete model of the effects of alloxan, the followng suggestons can be made. The recognton of glucose by B- cells s more rapdly and more severely affected by alloxan than the recognton of other secretagogues. However, after some delay, ther releasng effect s markedly mpared, wthout B-cells beng totally unresponsve, snce the decrease n K-permeablty s only margnally affected. It does not appear possble to explan these observatons by an acton of alloxan restrcted to a putatve membrane glucoreceptor. The possblty should be consdered that alloxan enters B-cells [33, 34] va the hexose carrer system where antagonsts of glucose transport (3-0-methylglucose, cytochalasn B, phlorzn) exert ther protectve acton [4, 17, 21, 34-36]. Alloxan would then nterfere wth cellular events crucal for the response to glucose and subsequently lead to a more general dysfunctonng of the releasng machnery. Acknowledgements. We are grateful to B. Debe, B. Moos and M. Nenqun for sklled techncal assstance, to M. Detalle and M. Nenqun for edtoral help and to Prof. D. P. Cameron for helpful dscussons. J. C. H. s "Charg6 de Recherches" from the Fonds Natonal de la Recherche Scentfque, Brussels. 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Res. Commun. 66, (1975) Receved: July 28, 1978, and n revsed form: October 17, 1978 Dr. J. C. Henqun Unt6 de Dab6te et Crossance Unversty of Louvan, UCL B-1200 Brussels Belgum