Glucose-induced changes in cytosolic ATP content in pancreatic islets

190

Biochimi~ etBiophvsica Acta 927 (1987) 190-195

Elsevier BBA 11876

G l u c o s e - i n d u c e d changes in cytosolic A T P c o n t e n t in pancreatic islets W i l l y J. M a l a i s s e a n d A b d u l l a h S e n e r Laboratory of Experimental Medicine, Brussels Free University, Brussels (Belgium)

(Received28 August 1986)

Key words: Adeninenucleotidecontent; Glucose;ATP; (Pancreaticislet)

The cytosolie and mitochondrial contents in ATP, ADP and AMP were measured in islets incubated for 45 min at increasing concentrations of D-glucose and then exposed for 20 s to digitonin. The latter treatment failed to affect the total islet A T P / A D P ratio and adenylate charge. D-Glucose caused a much greater increase in cytosolic than mitochondrial A T P / A D P ratio. In the cytosol, a sigmoidal pattern characterized the changes in A T P / A D P ratio at increasing concentrations of D-glucose. These findings are compatible with the view that cytosolic ATP participates in the coupling of metabolic to ionic events in the process of nutrient-induced insulin release.

Introduction The stimulant action of D-glucose and other nutrient secretagogues upon insulin release is causally linked to an increase in oxidative fluxes in pancreatic islet cells [1,2]. The coupling of metabolic to more distal events in the secretory sequence was proposed to represent a multifactorial process [3,4] including changes in the availability of reducing equivalents [5,6], protons [7,8] and high-energy phosphate intermediates [3,9]. Recent electro-physiological studies have revealed the presence in islet cell plasma membrane of K+-selective channels which are inhibited by ATP [10,11]. Hence, it is conceivable that the closing of such channels, in response to an increase in cytosolic ATP concentration, accounts for the nutrient-induced decrease in K + conductance, which is itself currently held responsible for the depolarization of the plasma membrane and subsequent gating of voltage-sensitive Ca2+-channels [12]. These considerations led us, in the present study, Correspondence: W.J. Malaisse, Laboratory of Experimental Medicine, Brussels Free University, Brussels B-1000, Belgium.

to examine the influence of increasing concentrations of D-glucose upon the cytosolic ATP content in rat pancreatic islets.

Materials and Methods In order to assess the effect of digitonin (Merck, Darmstadt, F.R.G.) upon the distribution of insulin, groups of 20 islets each, isolated by the collagenase method [13] from the pancreas of fed albino rats, were placed in small polythene tubes (Beckman Microfuge tubes) in 60/~1 of a glucosefree bicarbonate-buffered medium [13] containing bovine albumin (5 mg/ml). An aliquot (200/~1) of an iced imidazole-HC1 buffer (20 mM, pH 7.0) containing sucrose (250 mM), EDTA (3.0 mM) and digitonin (0.5 m g / m l , except if otherwise stated), freshly suspended by sonication, was then added to each tube. 20 s later, the tubes were centrifuged for 20 s at 5000 × g, and an aliquot (200 ~1) of the supernatant was removed. The pellet and remaining supernatant medium were mixed with 0.4 ml of a phosphate buffer (0.1 M, pH 7.0) containing bovine serum albumin (10 m g / m l ) and was sonicated (3 times for 10 s).

0167-4889/87/$03.50 © 1987 Elsevier SciencePublishers B.V. (Biomedical Division)

191 Prior to assay [13], all samples were further diluted in the same phosphate buffer in order to avoid the interference of digitonin with the immunoassay of insulin. The same procedure was used to assess the influence of digitonin upon the release of lactate dehydrogenase and glutamate dehydrogenase. Groups of 45 islets each were collected in the bicarbonate-buffered medium, washed twice in the same medium in order to minimize contamination by extracellular enzymes derived from the collagenase digestion, and eventually incubated for 45 min at 37°C in 30/~1 of the same medium. The volume of the imidazole buffer added to the incubation medium, and that of the supernatant removed after centrifugation and examined for its enzymatic content amounted to 100 #1. The enzymatic content of the supernatant was expressed relative to the total enzymatic content of the islets, which was measured, in each experiment, in parallel groups of 45 islets each sonicated (3 times for 10 s) in a medium of identical composition, i.e., a 0.3 : 1.0 (v/v) mixture of incubation medium and imidazole buffer. The activity of lactate dehydrogenase and glutamate dehydrogenase was measured as described elsewhere [14,15]. Essentially, the same procedure was used to measured the ATP, ADP and AMP content of either the cytosolic fraction (assumed to be released by digitonin) and mitochondrial fraction (assumed to be retained in the cell pellet after exposure to digitonin), except that each tube contained only 15 islets. After centrifugation in the Beckman Microfuge, an aliquot (100 #1) of the supernatant solution was rapidly mixed with 50/~1 of ice-cold HC104 (7.5%, v/v). Likewise, the pellet and remaining supernatant (30 /~1) were immediately mixed with 120 /tl of a mixture of incubation medium, imidazole buffer (see above) and HCIO 4 (13.4:44.8:41.8, v/v), so that the final composition of the cytosolic and mitochondrial extracts were identical to one another. The tube containing the islet pellet was then placed in liquid N 2, and the islets were homogenized by mechanical vibration at 400-800 cycles/s [16]. After a second centrifugation for 2 min in the Beckman Microfuge, aliquots (100/zl) of either the cytosolic or mitochondrial extracts were neutralized by addition of 50 ttl of a solution of KOH

(1.0 M) and Tris (0.2 M), and stored at - 6 0 ° C until they were used for the assay of adenine nucleotides. For the latter purpose, 3 aliquots of the extract (40 #1 each) were mixed with an equal volume of a Tris-HCl buffer (100 mM, pH 7.7) containing 100 mM KC1, 20 mM MgC12, 3 mM EDTA and 1.0 mM phosphoenolpyruvate (ATP assay). The reaction mixture also contained 25 mU of rabbit muscle pyruvate kinase (EC 2.7.1.40) for the combined assay of ATP and ADP and 140 mU of rabbit muscle myokinase (EC 2.7.4.3) for the combined assay of ATP, ADP and AMP. After 40 rain incubation at room temperature, the reaction was halted by heating for 5 min at 80°C. The samples were cooled and centrifuged. In all samples, ATP was measured in duplicate by the firefly lantern luciferase method [16,17]. Standards of ATP, ADP and ATP plus AMP were prepared in a medium of identical composition as the neutralized tissue extracts, and were treated in the same manner as the other samples. The readings obtained in the mitochondrial extract were corrected for contamination by the cytosolic material present in the supernatant solution (30 ~1) left on the top of the islet pellet, and the measurements made in each subcellular fraction were then related to the corresponding number of islets. All results are expressed as the mean value ( + S.E.) together with the number of individual observations (n). The statistical significance of differences between mean values was assessed by use of Student's t-test. Results

A first series of experiments was designed to assess the effect of digitonin upon the release of insulin, lactate dehydrogenase and glutamate dehydrogenase from the islet cells. As shown in Table I, only a minor fraction of insulin was released by the islets, even after exposure to a high concentration of digitonin. After 20 s exposure to digitonin, a maximal fractional release of lactate dehydrogenase was achieved with an imidazole buffer containing 0.5 m g / m l digitonin (Table I). At this concentration, the fractional release of glutamate dehydrogenase was much lower ( P < 0.001) than that of lactate dehydrogenase. The release of the mitochondrial enzyme tended to

192 TABLE I EFFECT OF DIGITONIN UPON THE RELEASE OF INSULIN, LACTATE D E H Y D R O G E N A S E A N D GLUTAMATE D E H Y D R O G E N A S E The concentration of digitonin refers to that of the imidazole buffer (0.1 ml) added to the incubation medium (0.03 ml) containing the islets. The release of insulin and enzymes over 20 s exposure to digitonin is expressed relative to the total islet content in insulin (1.18 _+0.08 mU/islet; n = 32), lactate dehydrogenase (100.8_+5.3 p m o l / m i n per islet; n = 30) and glutamate dehydrogenase (143.4_+14.5 p m o l / m i n per islet; n = 30). Digitonin (mg/ml)

Insulin (%)

0 0.1 0.5 5.0

1.1 _+0.3 (8) 0.3_+0.3 (8) 2.6_+0.3 (8) 3.1 _+0.8 (8)

Lactate dehydrogenase

Glutamate dehydrogenase

(%)

(%)

16.7_+0.9 (20) 58.5_+4.1 (19) 76.0_+3.4(23) 74.0_+4.8 (11)

5.1 _+0.5 (30) 13.0_+1.2 (19) 19.9_+1.5 (23) 27.9_+4.7 (11)

increase at higher concentrations of digitonin (e.g., 5.0 mg/ml). Exposure of the islet cells to digitonin (0.5 m g / m l or more) for more than 20 s also increased the release of glutamate dehydrogenase, but not that of lactate dehydrogenase. For instance, when the imidazole buffer contained 5.0 m g / m l dig±ton±n, the fractional release of lactate dehydrogenase averaged 77.2 + 2.9%, 70.9 _ 4.3%

TABLE II EFFECT OF DIGITONIN UPON THE RELEASE OF A D E N I N E NUCLEOTIDES, A T P / A D P RATIO A N D ADENYLATE C H A R G E The concentration of digitonin refers to that of the imidazole buffer (0.1 ml) added to the incubation medium (0.03 ml) containing the islets. The release of adenine nucleotides is expressed relative to the total islet content which averaged (expressed as pmol/islet) 2.24±0.08 for ATP, 0.83 ± 0.04 for ADP and 0.39+0.03 for AMP (n = 53 in each case). The A T P / A D P ratio and adenylate charge were calculated from the total ATP, ADP and AMP values (taken as the sum of released and retained nucleotides). Digitonin (mg/ml)

0

Release of adenine nucleotides ATP (%) 9.6 _+1.2 ADP(%) 6.1 +0.8 AMP (%) 2.2 _+1.0 ATP/ADPratio 2.83 +0.16 Adenylate charge 0.770 _+0.005

0.5 (25) (25) (25) (25) (25)

50.3 _+1.3 40.4 _+1.9 20.8 +3.1 2.89 _+0.17 0.768 ± 0,007

(28) (28) (28) (28) (28)

and 74.2 + 3.8% (n = 17 in each case) after 20, 40 and 60 s incubation, respectively. Under the same experimental conditions, the fractional release of glutamate dehydrogenase averaged 20.3 + 2.0%, 23.6 + 1.9% and 35.2 + 4.9% (n = 17 in each case) after 20, 40 and 60 s exposure to digitonin. In the light of these findings, all further experiments were performed with islets exposed for 20 s, prior to centrifugation, to an imidazole buffer containing 0.5 m g / m l digitonin. Table II illustrates the distribution of adenine nucleotides in control and dig±ton±n-treated islets. Digitonin significantly increased the release of ATP, ADP and AMP. Relative to the total islet content in each of these nucleotides, the dig±ton±n-stimulated fractional release of ATP was higher than that of ADP ( P < 0.001), which was itself much higher than that of AMP ( P < 0.001). Digitonin failed to affect either the A T P / A D P ratio or adenylate charge, which were both computed from the total islet content of each nucleotide (i.e., the amount of each nucleotide recovered in both the supernatant and islet pellet). The results given in Table II were actually collected in islets first incubated for 45 min either in the absence or presence of D-glucose. For the sake of simplicity, all results were pooled together. It was duly verified, however, that digitonin failed to affect the A T P / A D P ratio and adenylate charge whether the islets were first incubated with or without D-glucose (data not shown). In the next series of experiments, the effect of D-glucose upon the adenine nucleotide content of the islets was examined. Table III summarizes the data collected in islets incubated for 45 min in the absence or presence of D-glucose (16.7 mM) prior to being exposed to digitonin. Raising the D-glucose concentration significantly increased the ATP content of the cytosol ( P < 0.001), which represented a somewhat greater fraction of the total islet ATP content in glucose-stimulated than in glucose-deprived islets ( P <0.01). The cytosolic ADP content was decreased by D-glucose ( P < 0.001) when expressed as an absolute value (pmol/islet), but failed to be significantly affected by the hexose when expressed relative to the total ADP content. In the ghicose-deprived islets the cytosolic A T P / A D P ratio represented only 57.1 + 3.7% (d.f. = 100) of the mean value found within

193 TABLE III EFFECT OF D-GLUCOSE UPON THE CONCENTRATION AND DISTRIBUTION OF ADENINE NUCLEOTIDES D-Glucose (mM) a

0

Cytosolic values ATP (pmol/islet) ATP (%) b ADP (pmol/islet) ADP (%) b AMP (pmol/islet) AMP (%) b ATP/ADP (ratio)

1.16 50.4 0.58 41.4 0.07 15.3 2.16

Mitochondrial values ATP (pmol/islet) ADP (pmol/islet) AMP (pmol/islet) ATP/ADP (ratio) Adenylate charge (ratio)

16.7 _+0.04 (50) +1.0 (43) +0.02 (50) -+1.8 (35) -+0.02 (35) _+4.1 (35) _+0.09 (50)

1.08 _+0.04 (43) 0.67 +0.04 (35) 0.33 _+0.02 (35) 1.91 _+0.13 (35) 0.690 _+0.012 (28)

1.46 54.5 0.41 39.7 0.09 26.7 3.83

_+0.03 -+1.1 _+0.02 -+1.6 +0.01 _+4.4 _+0.17

(52) (44) (52) (35) (33) (33) (52)

1.17 +0.04 (44) 0.54 _+0.04 (35) 0.25 _+0.02 (33) 2.61 _+0.15 (35) 0.750 -+0.013 (27)

a The concentration of D-glucose refers to that of the incubation medium (0.03 ml). b The ATP, ADP and AMP values are expressed as percentages of the total islet content (taken as the sum of cytosolic and mitochondrial nucleotides).

the same experiments in glucose-stimulated islets. The increase in D-glucose concentration failed to affect significantly the cytosolic content in A M P (absolute value) which, however, tended to represent a greater fraction of the total islet content in glucose-stimulated than in glucose-deprived islets ( P < 0.06). I n the mitochondrial compartment, the rise in D-glucose concentration failed to affect significantly the A T P content ( P > 0.1), but decreased the A D P content by 16.7 _ 8.0% (d.f. = 68; P < 0.05) and the A M P content by 23.5 + 7.6% (d.f. --- 66; P < 0.005). The mitochondrial A T P / A D P ratio was increased in glucose-stimulated islets ( P < 0.005) but to a lesser extent ( P < 0.02) than the cytosolic A T P / A D P ratio. Indeed, in the mitochondria of glucose-deprived islets, the A T P / A D P ratio represented 76.3 + 7.7% (d.f. = 68) of the m e a n value f o u n d in the same experiments in glucose-stimulated islets (as distinct from 57.1 + 3.7% for the cytosolic A T P / A D P ratio; see above). The mitochondrial adenylate charge was also increased by D-glucose ( P < 0.005). W h e n both the cytosolic and mitochondrial A T P / A D P ratios were measured in the same samples, the latter averaged 83.3 + 6.0% (d.f. = 68; P < 0.01) of the former in glucose-deprived islets, as distinct ( P < 0.05) from only 64.7 _ 6.2% (d.f. = 68; P < 0.001)

in glucose-stimulated islets. All results so far presented refer to the situation f o u n d in glucose-deprived islets and those exposed to a high concentration of the hexose (16.7 mM). In the last series of experiments, we examined the effect of intermediate concentrations of D-glucose. The essential data are illustrated in Fig. 1. I n the cytosol, the A T P / A D P ratio increased as a function of the extracellular concentration of D-glucose. The relationship between these two variables followed a sigmoidal pattern. More precisely, a first increase in cytosolic A T P / A D P ratio was observed ( P < 0.001) when the concentration of D-glucose was raised from zero to 1.4 or 2.8 mM. A reascension in cytosolic A T P / A D P ratio was observed at higher concentrations of D-glucose in the 4.9 to 16.7 m M range. Both the absolute and relative magnitudes of the glucose-induced changes in A T P / A D P ratio were m u c h lower in the mitochondria than in the cytoplasm (Fig. 1). Although the mean values for the mitochondrial A T P / A D P ratio correlated with the corresponding D-glucose concentrations (r = 0.933; P < 0.01), only the difference between the m e a n basal value and that reached at the highest glucose level (16.7 m M ) achieved statistical significance ( P < 0.005).

194 4.0

(,,o)(22)(22) (50)

(28)

(2s)

(~2)

(,,)

(21)

(,,)

11'.1

16.7

3.5

cytosol ~,~_~

3.0

o (g k.

o.

Q <

2.5 o. I,<

2.0

(,9)(,,)(,,)

(3,)

61.a=

4',

1.-~ O:

D-glucose

(mM)

Fig. 1. Effect of increasing concentrations of D-glucose upon the cytosolic and mitochondrial A T P / A D P ratios. Mean values ( + S.E.) refer to the number of individual determinations quoted in parentheses.

Discussion

It is known from previous work that the ATP content, A T P / A D P ratio and adenylate charge are lower in islets deprived of exogenous nutrient than in islets exposed to D-glucose [1,18]. In these prior studies, the impression was gained that a concentration of D-glucose close to 2.8 mM may be sufficient to maintain the total islet content in ATP at the same level as that found in islets exposed to higher concentrations of the hexose.

However, the intracellular partition of adenine nucleotides had not been established in these previous experiments. In the present work, digitonin was used for the rapid separation of particulate components and soluble cytoplasm [19] in isolated rat islets. During the 20 s of exposure to digitonin, no significant change was detected in either the A T P / A D P ratio or adenylate charge, as computed from the total islet content in adenine nucleotides. As in other cell types [20,21], the A T P / A D P ratio was lower in the mitochondria than in the cytosol, such a difference being most marked when the rate of glycolysis was high, namely in islets exposed to elevated concentrations of D-glucose. Indeed, at increasing concentrations of D-glucose, the mitochondrial A T P / A D P ratio increased to a much lesser extent than the cytosolic A T P / A D P ratio. The results obtained in the mitochondrial compartment must be considered with caution, since they could reflect, to a limited extent, on adenine nucleotides located in secretory granules [221. The relationship between cytosolic A T P / A D P ratio and D-glucose concentrations displayed a sigmoidal pattern similar to that relating the metabolism of the hexose to its extracellular concentration [23]. This pattern contrasts with that characterizing the effect of increasing concentrations of D-glucose upon K + conductance, as judged from the changes in 86Rb outflow from prelabelled islets [24]. In the latter case, a closeto-maximal decrease in K + conductance is already observed at glucose concentrations well below those required to cause a maximal secretory response. Such a difference, however, does not rule out the view that cytosolic ATP participates in the control of K + conductance, since the response of K + channels to cytosolic ATP is not necessarily ruled by a law of strict proportionality [10,11]. In this respect, it could be objected that the data illustrated in Fig. 1 refer to the cytosolic A T P / ADP ratio, rather than ATP concentration. However, the cytosolic ATP content also increases a function of the extracellular concentration of Dglucose (Table III). It should also be underlined that, taking into account the intracellular space of each islet (approx. 2 nl), the cytosolie concentration of ATP lies in the millimolar range, whereas

195 the A T P - s e n s i t i v e K + channels close at microm o l a r c o n c e n t r a t i o n s of A T P [10,11]. In a d d i t i o n to their p o s t u l a t e d role in the control of K ÷ c o n d u c t a n c e , the changes in cytosolic A T P a n d A D P c o n c e n t r a t i o n s could c o n c e i v a b l y exert o t h e r r e g u l a t o r y influences. F o r instance, it was p r o p o s e d that m i t o c h o n d r i a l r e s p i r a t i o n is inversely related to the e x t r a m i t o c h o n d r i a l [ A T P ] / [ A D P ] - [ P i ] ratio [25,26]. However, in the islet cells, D-glucose increases O 2 c o n s u m p t i o n , despite the rise in the cytosolic [ A T P ] / [ A D P ] ratio. It seems unlikely that the g l u c o s e - i n d u c e d changes in cytosolic [Pi] a c c o u n t for such an a p p a r e n t discrepancy. Indeed, D-glucose lowers the islet orthop h o s p h a t e c o n t e n t [27], as a result of an increase in p h o s p h a t e efflux [28,29]. F o r instance, in the p r e s e n t system, the a m o u n t of Pi released b y i n t a c t islets in the extracellular m e d i u m , during 10 m i n i n c u b a t i o n at 3 7 ° C , increases ( P < 0 . 0 0 1 ) f r o m 4.6 + 0.8 to 11.4 _+ 0.8 p m o l / i s l e t (n = 13 in b o t h cases) when the c o n c e n t r a t i o n of D-glucose is raised f r o m zero to 16.7 m M . A l t h o u g h the c y t o solic c o n c e n t r a t i o n o f Pi r e m a i n s to be m e a s u r e d in the islet cells, the p r e s e n t findings are consistent with o t h e r c u r r e n t a r g u m e n t s a d v a n c e d against the view that m i t o c h o n d r i a l r e s p i r a t i o n d e p e n d s solely o n the [ A T P ] / [ A D P ] - [Pi] ratio [30,31]. In conclusion, the p r e s e n t w o r k reveals that, in p a n c r e a t i c islet cells, D-glucose causes a doser e l a t e d increase in cytosolic A T P c o n t e n t a n d cytosolic A T P / A D P ratio. These changes are c o m p a t i b l e with the view that cytosolic A T P acts as a c o u p l i n g factor b e t w e e n m e t a b o l i c a n d distal events in the process of n u t r i e n t - i n d u c e d insulin release, e.g., b y i n a c t i v a t i n g A T P - s e n s i t i v e K + channels.

Acknowledgements This w o r k was s u p p o r t e d b y a g r a n t from the Belgian F o u n d a t i o n for Scientific M e d i c a l Research. T h e a u t h o r s wish to t h a n k J. S c h o o n h e y d t a n d M. U r b a i n for technical assistance, a n d C. D e m e s m a e k e r for secretarial help.

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