Diabetologia 9 Springer-Verlag 1990

Transcription

1 Diabetlgia (1990) 33: Diabetlgia 9 Springer-Verlag 1990 mpairment f the mitchndrial xidative respnse t D-glucse in pancreatic islets frm adult rats injected with streptztcin during the nenatal perid M.-H. Girix, A. Sener 2, D. Bailbe 1, B. Prtha 1 and W. J. Malaisse e Labratire de Physilgie du D6velppement, Universit6 de Paris, Paris, France, and 2 Labratry f Experimental Medicine, Brussels Free University, Brussels, Belgium Smmnary. Pancreatic islets remved frm adult rats injected with streptztcin during the nenatal perid display an impaired secretry respnse t D-glucse and, t a lesser extent, t L-leucine. Despite nrmal t elevated hexkinase and gluckinase activities in the islets f these glucse-intlerant animals and despite nrmal mitchndrial binding f the hexkinase isenzymes, the metablic respnse t a high cncentratin f D-glucse is severely affected, especially in terms f D-[6-14C]glucse xidatin. Thus, the rati in D-[6-14C]glucse xidatin/d-[5-3h]glucse utilizatin is much less markedly increased in respnse t a rise in hexse cncentratin and, at a high cncentratin f D-glucse (16.7 mml/1), less markedly decreased by the absence f Ca 2t and presence f cyclheximide in diabetic than cntrl rats. This metablic defect cntrasts with (1) a clse-t-nrmal r even increased capacity f the islets f diabetic rats t xidize D-[6-14C]glucse, [2-~4C]pyruvate, L-[U-~4C]glu - tamine and L-[U-~4C]leucine at lw, nn-insulintrpic, cn- centratins f these substrates; (2) a lesser impairment f the xidatin f L-[U-14C]leucine tested in high cncentratin (20 mml/1), the effect f Ca 2+ deprivatin upn the latter variable being cmparable in diabetic and cntrl rats; (3) an unaltered transaminatin f either [2-~4C]pyruvate r L-[U- 14C]leucine; and (4) a mdest perturbatin f glyctysis. The mst bvius alteratin in glyclysis cnsists in a lesser increase f the glyclytic flux in respnse t a rise f D-glucse cncentratin in diabetic than cntrl rats, this cinciding with an apparent decrease in affinity f gluckinase fr the hexse. t is speculated that the preferential impairment f the metablic and secretry respnse t D-glucse may be mainly attributable t an altered cupling between calcium accumulatin and the stimulatin f xidative events in Beta-cell mitchndria f diabetic rats. Key wrds: Pancreatic islets, insulin release, streptztcin, glucse metablism, leucine metablism. The release f insulin evked by D-glucse in the pancreatic Beta cell is impaired in adult rats injected with streptztcin during the nenatal perid [1-3]. n this mdel f nn-insulin-dependent diabetes, the impairment f insulin secretin cincides with, and might be attributable t, a decrease in the xidative respnse f islet cells t a high cncentratin f the hexse [3]. This decrease in D-[U-14C]glucse xidatin and 02 cnsumptin cntrasts with a clse-t-nrmal rate f glyclysis [3]. The majr aims f the present study are t cmpare the xidatin f D-glucse and ther nutrients in the islets f diabetic and cntrl rats, t further explre the reciprcal cupling between functinal and mitchndrial xidative events in the islets islated frm either cntrl r diabetic animals, and t assess the pssible relevance f mitchndrial hexkinase isenzymes binding t the alteratin f the metablic islet respnse in diabetic rats. Materials and methds Cntrl rats and animals hajected with streptztcin [4] during the nenatal perids were given free access t fd [5] up t the time f killing. The rats were weighed and then decapitated, bld being cllected in heparinized tubes. After centrifugatin fr 10 min at 1000 g and 4 ~ the plasma was remved and stred at - 20 ~ The plasma glucse was measured by the glucse xidase methd [6] in 10 gl f plasma, and the plasma insulin in 100 gl f plasma using a radiimmunassay described elsewhere [7]. n each experiment, islets were islated by the cllagenase methd [8] frm the pancreases f three t fur rats. Tw grups f 15 islets each were snicated (3 x 10 s) in 0.25 ml H20 fr measurement f islet prtein by the methd f Lwry et al. [9] using bvine albumin as standard. An aliqut (25 gl) f the islet hmgenate was mixed with 1.0 ml f a phsphate buffer (100 mml/l, ph 7.0) cntaining bvine albumin (1.0%, weight/vlume) and stred at - 20 ~ fr measurement f the islet insulin cntent [8]. Fr measurement f insulin release, grups f eight islets each were incubated fr 120 min at 37 ~ in 1.0 ml f a bicarbnate-buf-

2 M.-H. Girix et al.: slet xidative respnse t D@ucse Table 1. Metablic status and islet functin Rats Cntrl Diabetic Age (weeks) 17.0_+ 0.1 (28) 13.1_+ 0.4 (28) a Bdy weight (g) 390 _+ 5 (28) 322 +_ 8 (28) ~ Plasma glucse (mml/1) 7.69 _ (26) (25) ~ Plasma insulin (muff) (25) (21) slet prtein (btg/islet) (16) (14) slet insulin (mu/islet) 1.58 _ (16) 0.39_ (16) a nsulin release (BU.islet min 1) - Nil 29.6_+ 6.5 (10) 20.4+_2.1 (28) - D-glucse (16.7 mml/1) (16) (31)" - L-leucine(20.0mml/1) 55.5+_ 5.9 (16) 34.0_+2.7 (31) a p < vs cntrl rats fered medium [8] cntaining bvine albumin (5 mg/ml) and equilibrated against a mixture f 02 (95%) and CO2 (5%). The amunt f insulin released in the media was measured as described elsewhere [8]. n all metablic studies, grups f 15 t 20 islets each were incubated fr 120 min at 37 ~ in 40 gl f the same bicarbnate-buffered medium cntaining, as required, bth D-[5-3H]glucse and D-[6-14C]glucse r nly a 14C-labelled nutrient. The incubatin was halted by additin f 20 gl f a citrate-naoh buffer (400 mml/1, ph 4.9) cntaining antimycin A (0.01 mml/1), rtenne (0.01 mml/1) and KCN (5.0mml/1). The xidatin f 14C-labelled nutrients was measured as described elsewhere [10], the 14CO2 being trapped in hyamine hydrxide ver 60 rain incubatin at rm temperature. When the medium als cntained D-[5-BH]glucse, the recvery f ~H20 in 0.5 ml f H20 was then cmpleted ver a further incubatin f 22 h at rm temperature [11]. The acidified incubatin media cntaining the islets were then stred at - 20 ~ and later used fr measurement f their cntent in either 14C-labelled amin acids, acidic metablites r lactate. Fr these measurements, the islets were disrupted by freezing and thawing. The 14C-labelled amin acids (e. g. alanine generated frm [2-14C]pyruvate) and acidic metablites (e. g. 2-ketiscaprate generated frm L-[UsaC]leucine) were separated frm their precursrs by chrmatgraphy n a Dwex 50 H + clumn, as previusly described [12]. Fr the assay f 14C-labelled lactate (generated frm D-[6-~4C]glucse) tw aliquts (40 ~tl each) taken frm distinct vials were pled tgether, heated fr 10 min at 70 ~ and neutralized by the additin f 15 btl NaOH (1.0 ml/1). Samples (40 tal each) f the neutralized extract were then mixedwith a reactin mixture and incubated with r withut lactate dehydrgenase fr determinatin f ~4C-labelled lactate, as described elsewhere [13]. Fr measuring the activity f hexkinase and gluckinase, grups f 400 t 600 islets were hmgenized in Ptter-Elvehjem tubes (20 strkes) in 0.3 ml f a Hepes-NaOH buffer (10 mml/1, ph7.0) cntaining 250mml/1 sucrse, 2mml/1 EDTA and 2 mml/1 L-cysteine. After 3 min centrifugatin at 1000 g in rder t remve nuclei, cell debris and intact cells, 0.25 ml f the supernatant was again centrifuged fr 60 min at 100,000 g and 4 ~ [14]. The supernatant f this secnd centrifugatin and the crrespnding pellet (resuspended in the same vlume f the same buffer) were bth snicated (3 x 10 s) and examined fr their glucse-phsphrylating activity, which was measured in aliquts f 10 t 30 gl, ver 60 min incubatin at 37 ~ in the presence f 2.0 mml/1 ATFP and in a final vlume f 60 gl, using a radiistpic prcedure previusly described [15]. Statistical analysis All results are expressed as the mean (_+ SEM) tgether with the number f individual determinatins (n), each cllected in a distinct rat r grup f islets, r degree f freedm (d. f.) and statistical significance f differences as assessed by use f Student's t-test. The SEM n the sum, difference r rati between mean values was calculated as indicated elsewhere [16, i7]. Results Metablic status and islet functin The bdy weight was lwer in diabetic than cntrl rats, but the frmer animals were als yunger than the latter nes (Table 1). When examined in the fed state, the plasma glucse cncentratin was higher in diabetic than cntrl rats, but n significant difference was fund in the plasma insulin cncentratin. The mean islet prtein cntent was smewhat lwer, but nt significantly s (p > 0.2), in diabetic than cntrl rats, whilst the insulin cntent f the islets was severely decreased in the diabetic animals. The basal insulin release, as measured ver 120 rain incubatin in the absence f exgenus nutrient, was nt significantly different (p > 0.05) in islets remved frm cntrl and diabetic rats, respectively (Table 1). n the presence f either D-glucse (16.7 mml/l) r L-leucine (20.0 mml/1), hwever, the secretry rate was lwer in diabetic than cntrl rats. The hexse-induced increment 655 Table 2. Oxidatin r utilizatin rate f nutrients by pancreatic islets Experiment 1 Labelled nutrient (mml/1) Ca 2+ (mml/1) Cntrl Diabetic D-[6-14C]glucse (2.8) 1.0 [2->C]pyruvate (1.0) e 1.0 L-[U-14C]glutamine (1.0) e 1.0 L-[U-14C]leucine (1.0) c 1.0 D-[5-BH]glucse (2.8) 1.0 D-[6-1aC]glucse (2.8) 1.0 D-[5-3H]glucse (16.7) 1.0 D-[6-14C]glucse (16,7) 1.0 D-[5-3H]glucse (16.7) Nil r D-[6-14C]glucse (16.7) NiF D-[6-14C]glucse (16.7) 1.0 L-[U-14C]leucine (20.0) 1.0 L-[U-14C]leucine (20.0) Nil e (pml- islet min - 1) _ 0.38 (15) (17) (15) (16) a (15) (17) (15) _ (17) b _+2.52(15) _+2.31(15) 4.08 _ (15) (15) _ (16) (16) d (16) _ 1.24 (16)" _ (15) (16) (15) _ 0.69 (16) _ (28) _ (25) a _ (28) _ (25) c (28) (25) p < 0.001; b p < 0.01; ~ p < 0.005; d p < 0.05 VS cntrl rats; e The incubatin media als cntained 2.8 mml/1 D-glucse; f The incubatin media als cntained EGTA (0.25 mml/1) and cyclheximide (0.05 mml/1)

3 656 Z._ 30 N O U e~ g m i Q 2G lo D -glucse N.S, P ~,001 N S, _y_ e,--,..1: 16.7 T 7-H 1 /.,1 /.e zd Ca Nil Fig.1. Rati f D-[6-~4C]glucse xidatin t D-[5-3H]glucse utilizatin in islets prepared frm cntrl (pen clumns) and diabetic (hatched clumns) rats and incubated in the presence f D-glucse (2.8 r 16.7 mml/1) with r withut Ca 2+. EGTA (0.25 mml/1) and cyclheximide (0.05 retl/l) wereincrprated in the Ca 2 +-deprived media. Mean values (+-SEM) refer t individual measurements, and are shwn tgether with the statistical significance (n. s.: nt significant) f differences between cntrl and diabetic rats in insulin utput was fur times higher (p < 0.001) in cntrl (111.7 _ gu/islet per 120 min) than diabetic rats ( ~tu/islet per 120 rain). The increment in insulin utput evked by L-leucine was nt significantly different (p>0.1), hwever, in cntrl ( gU/islet per 120 rain) and diabetic rats ( gu/islet per 120 rain). These data suggest that, in the diabetic rats, the secretry respnse t the amin acid was less severely impaired than that evked by the hexse. slet metablism When the xidatin f D-[6-14C]glucse, [2-14C]pyruvate, L-[U-14C]glutamine r L-[U-14C]leucine was measured in salt-balanced media and at lw, nn-insulintrpic, cncentratins f these nutrients, n significant impairment f z4co2 prductin was detected in the diabetic rats (Table 2, Expt. 1). On the cntrary, the nly significant changes cnsisted in a mdest increase in L-[U-14C]leu - cine xidatin and a mre marked increase in [2-~4C]pyru - vate xidatin. The hierarchy in the xidatin rate f distinct nutrients was nt identical in cntrl and diabetic rats. Fr instance, the rati between D-glucse and L-leucine xidatin was lwer (2<0.05) in diabetic ( %) than cntrl animals ( %). A different picture was btained when the metablism f D-glucse r L-leucine was examined at higher cncentratins f these nutrients. n the case f the hexse 16.7 M.-H. Girix et al.: slet xidative respnse t D-glucse (Table 2, Expt. 2), the rate f glyclysis, as judged thrugh the prductin f BH20 frm D-[5-3H]glucse, was nt significantly,different in diabetic and cntrl rats at a lw cncentratin f the hexse (2.8 mml/1). Hwever, at a higher cncentratin f D-glucse (16.7 mml/1), the rate f glyclysis was slightly lwer in diabetic than cntrl animals. Mrever, the 2.8/16.7 mml/1 rati in 3H20 prductin was significantly higher (p>0.01) in diabetic ( %) than cntrl rats ( %). n ther wrds, the rise in glucse cncentratin caused a lesser increase f glyclytic rate in diabetic than cntrl rats. Likewise, whereas the rate f D-[6-14C]glucse xidatin was nt significantly different in cntrl and diabetic rats at a lw cncentratin f the hexse, it was much lwer (p < 0.001) in the diabetic than cntrl animals at a high cncentratin f D-glucse. The rati between D-[6-14C]glucse xidatin and D-[5-BH]glucse utilizatin averaged, in cntrl and diabetic rats respectively, 13.2 _+ 0.7 and % in the presence f 2.8 retl/1 D-glucse and and % in the presence f 16.7 mml/1 D-glucse (Fig. 1). Thus, the rise in D-glucse cncentratin caused a preferential stimulatin f the xidatin f acetyl residues in the Krebs cycle, relative t the rate f glyclysis, in bth cntrl and diabetic rats (p < 0.005). Hwever, the relative magnitude f such a preferential stimulatin was lwer (p < 0.02) in diabetic ( %) than cntrl rats ( _ 10.6%). n rder t gain further insight int the significance f these metablic changes, the experiments cnducted in the presence f 16.7 mml/1 D-glucse were repeated in media deprived f CaCla and enriched with bth EGTA (0.25 retl/l) and cyclheximide (0.05 mml/1). Under the latter experimental cnditins, which aimed at suppressing the functinal respnse f islet cells t D-glucse [11], the rate f D-[5-gH]glucse utilizatin was nt significantly affected, whether in cntrl r diabetic animals. The absence f Ca 2+ and presence f cyclheximide significantly decreased (p < 0.005), hwever, the rate f D-[6J4C]glucse xidatin, in bth cntrl and diabetic rats. The rati between D-[6-t4C]glucse xidatin and D-[5-gH]glucse utilizatin was als markedly decreased, in bth cntrl and diabetic rats (Fig. 1). Once again, the relative magnitude f the latter change was mre prnunced (p < 0.025) in cntrl ( %) than diabetic rats ( %). The study f D-[6-14C]glucse catablism by intact islets als included the measurement f 14C-labelled lactate prductin. Expressed as pml f glucse residue/islet per 120 rain, it averaged, in cntrl and diabetic rats respectively, (n = 15) and (n = 14) in the presence f 2.8 mml/1 D-glucse, (n = 18) and (n = 18) in the presence f 16.7 mml/1 D-glucse, and (n = 8) and (n = 7) at the same high cncentratin f the hexse but in the absence f Ca 2+ and presence f EGTA (0.25 retl/l) and cyclheximide (0.05 mml/1). These data indicate that the prductin f lactate was nt significantly different in cntrl and diabetic rats, whether at a lw (2.8 mml/1) r high (16.7 mml/1) cncentratin f D-glucse. When measured within the same experiments, the rati f

4 M.-H. Girix et al.: slet xidative respnse t D-glucse Table 3. Transaminatin f nutrients in pancreatic islets Experiment (as in Table 2) Labelled nutrient (mml/1) Ca 2+ (mml/1) Cntrl Diabetic (pml. islet min 7) 1 [2-14C]pyruvate(1.0) b _ (15) (17) L-[U-14C]leucine (1.0) b _ 3.29 (15) 45.09_ (17) a 3 L-[UJ4C]leucine (20.0) (28) _ 7.19 (25) L-[U-14C]leucine (20.0) Nil c (28) 98.74_ (25) p < 0.001; b The incubatin media als cntained 2.8 mml/1 D-glucse; c The incubatin media als cntained EGTA (0.25 mml/1) and cyclheximide (0.05 mml/1) Table 4. Hexkinase activity in islet hmgenates Rats Cntrl Diabetic Subcellular fractin Cytsl Mitchndria Cytsl Mitchndria D-glucse phsphrylatin rate (pml - islet - 1. rain- ~) - N G6P - G6P (3.0 retl/l) Glucse 6-phsphate-resistant activity (%) Fractinal cntributin f each subcellular fractin (%) _ 0.06" _ _ _ _ _ _ _ N G6P 55.2 _ _ _+ 2.6 Mean values ( + SEM) are derived frm six individual measurements in all cases 657 [~4C]lactate prductin at 16.7/2.8 mml/1 D-glucse was significantly lwer (p<0.005), hwever, in diabetic ( ) than cntrl rats (4.37 _+ 0.58), as already bserved in the case f D-[5-3H]glucse utilizatin (see abve). The absence f Ca 2 + and presence f cyclheximide failed t affect significantly the prductin f [14C]lactate, the trend being twards a decreased generatin f [~4C]lactate in bth cntrl and diabetic rats. n bth cntrl and diabetic rats, the rate f aerbic glyclysis, taken as the difference between the prductin f 3H20 frm D-[5-3H]glucse and the generatin f [14C]labelled lactate, increased significantly (p > 0.001) in respnse t a rise in D-glucse cncentratin frm 2.8 t 16.7 mml/1. Mrever, when expressed relative t the crrespnding ttal glyclytic flux, the fractinal cntributin f aerbic glyclysis als increased as the cncentratin f D-glucse was raised frm 2.8 t 16.7 mml/1. The magnitude f such a change was cmparable in nrmal and diabetic rats and, pling all available data cllected in bth types f rats, crrespnded t an increase in the fractinal cntributin f aerbic t ttal glyclysis frm % in the presence f 2.8 mml/1 D-glucse t % in the presence f 16.7 mml/1 D-glucse (p < 0.02). The last experiments in this series aimed at cmparing the xidatin f D-.[6-~4C]glucse and L-[U-~4C]leucine (Table 2, Expt. 3). The xidatin f L-[U-~4C]leucine, at a high cncentratin f the amin acid (20.0 mml/1) and in salt-balanced media, was lwer in diabetic than cntrl rats. When measured within the same experiments, the xidatin rate f D--[6-~4C]glucse (16.7 mml/1) relative t that f L-[U-14C]leucine (20.0mml/1) averaged % (n -- 28) in cntrl rats, as distinct (p < 0.05) frm nly % (n = 25) in diabetic rats. This indicates a mre severe alteratin f the mitchndrial xida- tin f the hexse rather than amin acid, as a result f the nenatal administratin f streptztcin. Mrever, in the absence f CaC12 but presence f EGTA (0.25 mml/1) and cyclheximide (0.05 mml/1), the xidatin f L-[U-14C]leucine (20.0 mml/1) was decreased (p<0.005) t % (d.f.=54) and % (d. f. = 48) f its crrespnding cntrl value in nrmal and diabetic rats, respectively. The latter tw percentages were nt significantly different frm ne anther (p > 0.5), in sharp cntrast t the situatin fund with D-[6-14C]glucse (16.7 mml/1). This indicates that, in terms f the interdependency between xidative and functinal respnses, the situatin in diabetic animals was again mre severely perturbed in glucse- than leucinestimulated islets. The peratin f transaminatin reactins in intact islets was als investigated by measuring the generatin f 14C-labelled amin acids in islets expsed t [2-~4C]pyru - vate and that f 14C-labelled acidic metablites in islets expsed t L-[U-14C]leucine (Table 3). The generatin f 14C-labelled amin acids, presumably mainly [2- ~4C]alanine, by islets expsed t [2-14C]pyruvate was nt significantly different in nrmal and diabetic rats. The generatin f lgc-labelled acidic metablites, mainly [U- 14C]2-ketiscaprate [18], by islets expsed t L-[U- ~4C]leucine was higher (p < 0.001) in diabetic than cntrl rats, when the amin acid was tested at a lw cncentratin (1.0 mml/1). Such a difference was n lnger bserved, hwever, when L-leucine was tested at a cncentratin f 20.0 mml/1. n the latter case, the absence f Ca 2 + and presence f cyclheximide decreased (p < 0.005) the generatin f ~4C-labelled acidic metablites in bth cntrl and diabetic rats. The relative extent f such an inhibitin was smewhat less marked, albeit nt significantly s, in diabetic rats ( %; d. f. = 48) than cn-

5 658 u 0 => Z _.Q p.. 0 P. U~ "" et uj 0q (J Q ,5 i P<~01 CYTOSOL 1 1,,A,A 'A p <~3 l PELLET Fig. 2. Effect f a rise in the cncentratin f unlabelled D-glucse frm 10 t 50 mml/l upn the phsphrylatin rate f D-[6-14C]glucse by islet subcellular fractins prepared frm cntrl (pen clumns) and diabetic rats (hatched clumn). Mean values ( _+ SEM) refer t six individual measurements and are expressed relative t the mean reference value fund, within the same experiment, in the cntrl rats. Such a reference value crrespnded t a furt five-fld decrease in the generatin f D-[6-14C]glucse 6-phsphate trl animals ( %; d.f. = 54). n bth cntrl and diabetic rats, the xidatin f L-[U-14C]leucine relative t the generatin f [U-~4C]2-ketiscaprate and ther 14Clabelled acidic metablites was higher in the presence f 1.0 retl/1 rather than 20.0 retl/1 L-leucine. Thus, such a relative xidatin rate decreased, in respnse t the rise in L-leucine cncentratin, frm t % in cntrl rats and frm t % in diabetic rats (p < 0.005). D-glucse phsphrylatin by islet hmgenates Because f the preferential perturbatin f D-glucse catablism in the islets f diabetic rats and in view f the essential rle currently ascribed t D-glucse phsphrylatin in the regulatin f hexse metablism in islet cells, the activity f hexkinase and gluckinase in islet hmgenates was examined in the last part f the present M.-H. Girix et al.: slet xidative respnse t D-glucse study. Mrever, because f the ptential regulatry rle f hexkinase isenzymes binding t mitchndria, the enzymatic data were cllected in bth a particulate and sluble subcellular fractins prepared frm the islet crude hmgenates. The ttal activity f hexkinase, as measured in the presence f 0.6 mml/1 D-glucse, was higher in diabetic than cntrl rats, averaging respectively and pml/islet per rain (p < 0.005). The relative cntributin f cytslic and mitchndrial material t the ttal rate f hexse phsphrylatin was nt significantly different in cntrl and diabetic rats (Table 4). D- glucse 6-phsphate (initial cncentratin: 3.0 mml/l) severely inhibited hexkinase activity. n bth cntrl and diabetic rats, the relative extent f such an inhibitin was higher (p < 0.001) in the cytslic than mitchndrial material, as expected frm prir bservatins [14, 19]. The ttal activity f gluckinase, as judged frm the paired difference in the phsphrylatin rate f D-glucse tested at cncentratins f 0.6mml/1 and 10.0 mml/1 respectively, was nt significantly different (p > 0.2) in cntrl ( pml/islet per min) and diabetic rats ( pml/islet per rain). t accunted, in cntrl and diabetic animals respectively, fr % and % f the ttal rate f hexse phsphrylatin recrded in the presence f 10.0 mml/1 D-glucse (Table 5). The fractinal cntributins f the sluble and particulate-bund gluckinase were similar in cntrl and diabetic rats, accunting each fr abut half f the ttal enzymatic activity. n the study f gluckinase activity, the sle difference between cntrl and diabetic rats was bserved when the cncentratin f unlabelled D-glucse was raised frm 10 t 50 mml/1, whilst keeping the cncentratin f D-[6- ~4C]glucse unchanged. The decrease in the generatin rate f D-[6-14C]glucse 6-phsphate resulting frm such an increase in the cncentratin f unlabelled D-glucse was indeed less marked in diabetic than cntrl rats (Fig. 2). This difference was bserved in bth the cytslic and mitchndrial fractin. Discussin The present study extends prir investigatins n the metablic and functinal anmalies in islets prepared frm adult rats which were injected with streptztcin during Table 5. Gluckinase activity in islet hmgenates Rats Subcellular fractin D_glucse phsphrylatin rate (pml. islet- 1. min 1) Cntrl Cytsl 0.69 _ a Fractinal cntributin t the ttal hexse phsphrylatin rate at 10 mmi/1 D-glucse (%) Fractinal cntributin f each subcellular fractin (%) 49.4 _ " Mean values ( _+ SEM) are derived frm six individual measurements in all cases Diabetic Mitchndria Cytsl Mitchndria

6 M.-H. Girix et al.: slet xidative respnse t D-glucse the nenatal perid [2, 3]. These anmalies may result frm a lng-lasting effect f streptztcin [19] and/r mild hyperglycaemia. Our results suggest that the capacity f mitchndria t xidize D-glucse, pyruvate, L-glutamine and L-leucine is nt impaired in the diabetic rats, at least when these nutrients are tested at nn-insulintrpic cncentratins and even mre s that the size f the islets might be smewhat lwer in diabetic than cntrl rats. Likewise, the transaminatin data cllected in intact islets indicate that there is n alteratin f the latter prcess in diabetic rats. Since the generatin f 2-ketiscaprate frm L-leucine, in the islets, is critically dependent n the activatin f glutamate dehydrgenase by the amin acid [20], these data als suggest that there is n bvius perturbatin in the activity f the latter mitchndrial enzyme. Nevertheless, the fact that, in the presence f 20.0 mml/1 L-leucine, the xidatin f the amin acid was lwer in diabetic than cntrl rats pints t an alteratin in the xidative catablism f 2-ketiscaprate, generated frm exgenus L-leucine, as a pssible cause f the impaired secretry respnse t L-leucine recrded in the islets frm diabetic rats. The latter secretry defect sharply cntrasts with the increased respnsiveness t L-leucine recrded in the perfused pancreas f diabetic rats [1]. The factrs respnsible fr such cntrasting behaviur f the Beta cell in the perfused pancreas and islated islets, respectively, remain t be identified. Three cnverging series f bservatins indicated that the metablic and secretry respnse f islet cells was mre severely perturbed in the case f D-glucse than L-leucine. First, as judged frm the increment in insulin release evked by these nutrients, the respnse t D-glucse was mre markedly affected than that t L-teucine. Secnd, a preferential alteratin f D-glucse catablism was dcumented by the direct cmparisn f D-[6- ~4C]glucse and L-[U-14C]leucine xidatin in bth cntrl and diabetic rats. n this respect, cmparable results were btained at lw and high cncentratins f these nutrient secretaggues. Third, the reciprcal cupling between xidative and functinal events, as judged frm the changes evked by the absence f Ca 2+ and presence f cyclheximide [11], als indicated a severe alteratin in the case f glucse-stimulated islets prepared frm diabetic rats, whereas n significant difference between nrmal and diabetic rats was recrded when the same envirnmental factrs were tested in leucine-stimulated islets. The present results suggest that the preferential alteratin f the Beta cell respnse t D-glucse in this mdel f nn-insulin-dependent diabetes is nt attributable t either a decrease in hexkinase and gluckinase activities r an altered binding f these isenzymes t mitchndria. n the case f the high Km gluckinase, the sle perturbatin in the diabetic animals resided in an apparently decreased affinity fr D-glucse. The same change in the kinetic behaviur f gluckinase was recently bserved in the liver f streptztcin-induced diabetic adult rats [21]. This anmaly might pssibly accunt, in part at least, fr the lesser increase in glyclytic flux bserved in the islets f diabetic rather than cntrl rats in respnse t a rise in 659 extracellular D-glucse cncentratin frm 2.8 t 16.7 mml/1. The abslute rate f glyclysis at the high cncentratin f D-glucse was little affected, hwever, in the islets f diabetic rats. The majr metablic anmaly in glucsestimulated islets frm diabetic rats cnsisted in an impaired preferential stimulatin f D-[6-14C]glucse xidatin. This mitchndrial defect cincided with a lesser sensitivity twards the absence f Ca 2 and presence f cyclheximide f the same bichemical prcess, i.e. the xidatin f acetyl residues in the Krebs cycle in islets expsed t a high cncentratin f extracellular D-glucse. Recent bservatins ascribe a key rle t the mitchndrial accumulatin f calcium and subsequent activatin f calcium-dependent mitchndrial dehydrgenases (e. g. glycerl phsphate dehydrgenase and 2-ketglutarate dehydrgenase) in the preferential stimulatin by D-glucse f mitchndrial xidative events (such as circulatin in the glycerl phsphate shuttle and acetyl residues xidatin in the Krebs cycle) in the islet cells [22, 23]. t is cnceivable, therefre, that the altered respnse f pancreatic islets t either a rise in D-glucse cncentratin r the remval f Ca 2+, as judged frm the rati between D-[6-14C]glucse xidatin and D-[5-3H]glucse utilizatin, is mainly attributable t an alteratin, at the mitchndrial level, f this reciprcal cupling between catinic and xidative events. n cnclusin, althugh the perturbatin f the Betacell secretry behaviur in the present mdel f nn-insulin-dependent diabetes might be attributable t several distinct metablic defects, ur results pint, nevertheless, twards an alteratin in the interdependency f Ca 2 handling and mitchndrial xidative events as a majr determinant f the preferential impairment f the Betacell respnsiveness t D-glucse. n this respect, the present findings emphasize the view [24] that the transprt f D-glucse acrss the plasma membrane and its subsequent phsphrylatin by gluckinase shuld nt be cnsidered as the sle, pssibly even nt the majr, regulatry factrs f the functinal respnse t D-glucse, whether in nrmal r diseased Beta cells. Acknwledgements. The authrs are mst grateful t J. Schnheydt and M. Urbain fr technical assistance and C. Demesmaeker fr secretarial help. This wrk was supprted by grants frm the nstitut Natinal de la Santd et de la Recherche Mddicale (France), Fundatin fr Scientific Medical Research (Belgium) and Ministry f Scientific Plicy (Belgium). References 1. Girix M-H, Prtha B, Kergat M, Bailbe D, Picn L (1983) Glucse insensitivity and aminacid hypersensitivity f insulin release in rats with nn-insulin-dependent diabetes: a study with the perfused pancreas. Diabetes 32: Prtha B (1985) Decreased glucse-induced insulin release and bisynthesis by islets f rats with nn-insulin-dependent diabetes: effect f tissue cultm-e. Endcrinlgy 117: Prtha B, Girix M-H, Serradas R Welsh N, Hellerstr0m C, Sener A, Malaisse WJ (1988) nsulin prductin and glucse metablism in islated pancreatic islets f rats with nn-insulin-dependent diabetes. Diabetes 37:

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