Effect of chloroquine on biosynthesis, release and degradation of insulin in isolated islets of rat pancreas

Diabetes Research and Clinical Practk'e, 5 (1988) 9-15 Elsevier DRC 00196

Effect of chloroquine on biosynthesis, release and degradation of insulin in isolated islets of rat pancreas A.K. Chatterjee* and H. Schatz IH Medical Department, Centre ~)f hlternal Medicine, Universi O' of Giessen, D-6300 Giessen, F.R.G. (Received 12 December 1986, revision received 6 November 1987, accepted 3 December 1987)

Key words: Chloroquine; Islet culture; (Pro)insulin biosynthesis: Insulin release; Insulin degradation

Summary Insulin has been reported to degrade inside the islets and islet lysosomal proteases have been thought to take part. As chloroquine is regarded as a potent lysosomotropic agent, an attempt has been made to see whether chloroquine has an influence on intrainsular degradation of insulin. After preculture of collagenase-isolated rat islets at 11 mM glucose together with [3H]leucine for 3 days for labelling newly synthesized insulin, islets were cultured for 1 day at 2.2 or 22 mM glucose with or without 0.02 mM chloroquine. Afterwards, radioactivity was measured in the proinsulin/insulin fraction. For control, the influence of chloroquine during 3-h incubation of both freshly isolated and precultured islets was also studied. During the 1-day culture at 2.2 mM glucose, prelabelled insulin was degraded significantly and addition of chloroquine did not alter the amount of insulin degraded. At 22 mM glucose, no significant amount of insulin had been degraded. During the 3-h incubation of freshly isolated as well as precultured islets, chloroquine was found to inhibit significantly glucose-induced biosynthesis of insulin. Glucose-induced release of insulin, however, was not influenced by chloroquine. It is concluded that chloroquine does not influence glucose-induced release or intra-insular degradation of insulin, but it interferes with the biosynthesis of insulin.

Introduction

Address for correspondence: Dr. H. Schatz, 111 Medical Department, Ceqtre of Internal Medicine, University of Giessen, D-6300 Giessen, F.R.G. * Present address: Center for Environmental Toxicology, Michigan State University, C-231 Holden Hall, East Lansing, Michigan 48824-1206, U.S.A.

As in other endocrine cells, insulin is known to be degraded inside the pancreatic islets, and such intra-insular degradation of insulin has been thought to be regulated by insulin release as well as the glucose concentration per se [1-3]. However, irrespective of the regulation mechanism, until now very

0168-8227/88/$03.50 C~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

10 little has been known regarding the mechanism involved. Lysosomal [4] or microsomal [5,6] enzymes were believed to be the most fitting candidates for insulin degradation and recently Schnell et al. [7] have immunocytochemically demonstrated the presence of insulin in pancreatic islet lysosomes. As chloroquine is regarded as a potent lysosomotropic agent [8], in the present study an attempt has been made to see whether chloroquine influences intrainsular degradation of preformed insulin by changing tile activities of islet lysosomal proteases. Concomitantly, the effect of chloroquine on the biosynthesis and release of insulin was also studied.

Materials and methods

MateriaLs' Collagenase was purchased fl'om Serva, Heidelberg (F.R.G.), glucose-free medium RPMI 1640 and newborn call" serum were obtained from Gibco, Glasgow (U.K.); t,-[4,5-3H]leucine (45 Ci/mmol) from the Radiochemical Centre, Amersham (U.K.), and Soluene-350 (tissue solubiliser) and Instafluor from United Technologies, Packard (U.K.). Bovine anti-insulin serum (guinea-pig) was obtained from Miles-Yeda, Rehovot (Israel) and normal guineapig serum from Miles Lab., Kankakee, IL (U.S.A.). Nonidet P-40 was purchased fi'om Fluka AG, Buchs (Switzerland), petri dishes (diameter 5 cm) were from A/S NUNC, Roskilde (Denmark) and rat insulin from Novo Research Lab., Copenhagen (Denmark). Chloroquine diphosphate was purchased from Sigma Chemical Co. (U.S.A.); penicillin and streptomycin from Boehringer, Mannheim (F.R.G.) and the insulin radioimmunoassay kit from Behringwerke, Marburg/Lahn (F.R.G.). Other reagents were of analytical grade. Animals and islet isolation Male Wistar rats (b.w. 150 200 g) fed ad libitum were used in the experiments. To improve yield of islets, 0.5 ml of 2% pilocarpine hydrochloride solution was injected i.p. 2 h before decapitation of the rats [9]. Pancreatic islets were isolated aseptically by the collagenase rnethod [10]. Krebs Ringer

bicarbonate (KRB) buffer, pH 7.4, containing aprotinin (800 kIU/ml) and glucose (2.8 raM) was used throughout the procedure. Islet culture and hwuhation procedure Batches of 50 islets were transferred to petri dishes containing 5 ml of medium RPMI 1640, supplemented with I I mM glucose, 1 ml of heat-inactivated (56°C) newborn calf serum, 10000 U/I penicillin and 100 U/1 streptomycin. They were cultured for 3 days in an incubator (37°C, 5% CO2) under sterile conditions. At the end of the 3-day culture period, the islets were washed three times in KRB. Groups of 20 freshly isolated or the precultured (for 3 days) islets were transferred to test tubes containing 1 ml of KRB, supplemented with 17 naturally occurring amino acids (20 iLg/ml of each amino acid, leucine excluded), bovine serum albumin (2 mg/ml), pH]leucine (20 iLCi/ml) and either 2.2 or 22 mM glucose with or without 0.02 mM chloroquine. The islets were then incubated for 3 h at 37°C in a metabolic shaker under a constant atmosphere of O2:CO==95:5% with gentle shaking (90 cycles/ rain). Aliquots of the medium were collected at hourly intervals for measuring release of immunoreactive insulin, using rat insulin as the standard. At the end of the 3-h incubation period, aliquots were collected also for measuring release of newly synthesised (pro)insulin in the medium (see below). When incubation was over, the islets were washed twice in phosphate-buffered saline (PBS, pH 7.4) containing an excess of unlabelled L-leucine (20 raM), and frozen in 1 ml of PBS. °,

Determination o/ degrcukttion or produclion ~!/" hlsulin Batches of islets were precultured as above for 3 days with supplementation of 20 llCi/ml [3H]leucine in the medium for the purpose of prelabelling the newly synthesised (pro)insulin during the culture period. The islets were then washed three times in KRB. For estimation of the content of tritiated (pro)insulin directly after the labelling period, part of the islets were transferred to test tubes, washed twice in PBS and ['rozen in I ml PBS. The remaining

11 islets were further cultured for 1 day (degradation period) in medium (without [3H]leucine) containing either 2.2 or 22 mM glucose with and without the addition of 0.02 mM chloroquine. At the end, the islets were similarly washed in PBS and frozen in 1 ml of PBS. Aliquots of the media were also frozen. The amount of labelled (pro)insulin m the islets frozen directly after the labelling period and in the islets and in lnedia collected after the degradation period was measured after immunoprecipitation and binding to protein A-Sepharose CL 4B (see below). The radioactivity of (pro)insulin found in the islets following prelabelling was considered as the 'initial wdue'. The difference between this value and the sum of the radioactivity of (pro)insulin measured in the islets and media after the degradation period represented a measure for intra-insular degradation or production of insulin.

Delerm#ullion o/" &hefted (l~ro )hzsulin After thawing, the islets were sonified for 15 s at 2-4°C. Newly formed (pro)insulin (in terms of [3H]leucine incorporation)in aliquots of each batch of the sonified islets and corresponding incubation media was estimated, using CNBr-activated protein A-Sepharose CL 4B and anti-bovine insulin antibodies. The procedure was essentially the same as described earlier [2] except for the following changes: the antigen-antibody complex from Sepharose beads was solubilised overnight using 0.5 ml of Soluene, and after centrifugation a fraction of 0.3 ml fl'om the supernatant was transferred to scintillation vials and then 10 ml of Instafluor was added for measuring the radioactivity in a betacounter.

Data aua/l'.s'L~"

Mean and SEM of each set of data were calculated and statistical ewduations were made using Student's t-test. Results Following prelabelling for 3 days, 1163 + 92 cpm/islet (X 4- SEM, n = 6) was determined as the

"initial value" for the tritiated (pro)insulin content of the islets. After cultivation for an additional 24 h in tile presence of 2.2 mM glucose without labelled amino acid, approximately 51% of this value could be recovered in islets plus medium. However, after cultiwltion at 22 triM glucose, almost 75% of the "initial wdue' was recovered and the difl'erence fl'om the 'initial value" was calculated to be insignificant (Fig. 1). Fig. 1 also shows that at both concentrations of glucose, addition of 0.02 mM chloroquine did not alter the corresponding values significantly. But, as compared to the control values, in the presence of 2.2 mM glucose, addition of chloroquine caused an increased release of labelled (pro)insulin (2.2 mM glucose: 14 4- 3 cpm/islet: 2.2 mM glucose + chloroquine: 87 + 21 cpm/islet: P<0.01). On the other hand, in the presence of 22 mM glucose, chloroquine caused an inhibition of such release (22 mM ghtcose: 369 4- 59 cpm/islet: 22 mM glucose + chloroquine: 176 4- 59 cpm/islet: P < 0.02). As listed in Table 1, in the presence of 2.2 mM glucose, the freshly isolated and 3-day precultured islets synthesised almost the same arnount of (pro)insulin during the 3-h static incubation period. However, in the presence of 22 mM glucose, the 3-day precultured islets synthesised a decreased O =

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Fig. I. El'feet of chloroquinc diphosphatc on the degradation of prelabelled (pro)insulin in rat islets during 24 h of cultivation at 2.2 or 22 mM glucose. Left: a m o u n t of radioactive (pro)insulin prcsenl in the islets after prelabelling I\+r 3 days in culture tit I I mM glucose together with [-+llJleucine. Middle and right bars: amount of radioactive (pro)insulin still present in islels (open bars) and in media (closed bars) after an addilional 24 h of cultivation in cold medium containing 2.2 or 22 mM glucose with and without the addition of 0.02 mM of chloroqume diphosphale. Values represent mean :k SEM of six individual observations with slalistical signilicance (P). n.s. = not signilicant.

12 TABLE I E F F E C T O F C H L O R O Q U I N E DIPHOSPI-IATE ON T H E G L U C O S E - I N D U C E D B I O S Y N T H E S I S O F ( P R O ) I N S U L I N D U R ING 3-h I N C U B A T I O N O F F R E S H L Y I S O L A T E D O R P R E C U L T U R E D ( F O R 3 D A Y S A T I 1 m M G L U C O S E ) R A T PANC R E A T I C ISLETS Individual data refer to the sum total of labelled (pro)insulin estimated in the islet at the third hour of incubation as well as that released in the incubation medium. Values are mean 4- SEM with statistical significance (P). NS = not significant, n = n u m b e r of individual observations. Chloroquine (raM)

Glucose 2.2 rnM Fresh islet (cpm/islet) n= 9

Glucose 22 m M Precultured islet (cpm/islet) n= 6

Fresh islet (cpm/islet) n= 9

Precultured islet (cpm/islet) n= 6

NS 0 0.02 P

219 + 67 95 + 17 < 0.05

210 ± 27 112 • 24 < 0.025

amount of (pro)insulin compared to that synthesised by the freshly isolated islets: but the difference was found to be statistically insignificant. However, during the 3-h incubation of the freshly isolated as well as precultivated islets, chloroquine caused a significant inhibition of glucose-induced synthesis of (pro)insulin except in the case of the fresh islets in the presence of 22 rnM glucose. But, as shown in Fig. 2 (left upper and lower panels), chloroquine did not significantly alter the glucoseinduced (either 2.2 or 22 raM) release of insulin during the 3-h incubation of the freshly isolated islets. Although during the incubation of the precultured islets at 2.2 mM glucose chloroquine caused an increase in the release of imrnunoreactive insulin, this was not found to be statistically significant as compared to the control value (2.2 mM glucose: 11.6 + 2.6 /tU/islet; 2.2 mM glucose + chloroquine: 18.6 -4- 7.6l~U/islet: NS)(right tipper panel). During incubation of the precultured islets at 22 mM glucose, chloroquine did not cause any alteration in the release of immttnoreactive insulin (right lower panel). It is clear from Fig. 2 that altogether, at both concentrations of glucose, the freshly isolated islets released a larger amount of immunoreactive insulin than the precultured islets.

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Fig. 2. Effect orchloroqumc diphosphate on the glucose-reduced release of immunoreactive insulin during 3-h incubation of freshly isolated or precultured (for 3 days at 11 m M glucose) rat pancreatic islets. Left: release of insulin from freshly isolated islets during 3-h incubation lit 2.2 rnM glucose (upper panel) or 22 mM glucose (lower panel) with or without the addition of 0.02 m M chloroquine diphosphate. Right upper and lower panels: results of similar studies with islets precultivated for 3 days at 11 m M glucose. Values represent mean ± SEM. Figures in parentheses indicate the n u m b e r of individual observations.

13 Discussion

In agreement with the earlier observations [1-3], the present results demonstrate that at a low glucose concentration (2.2 mM), intra-insular insulin was degraded significantly, which was not the case in the presence of a high glucose concentration (22 raM). Clearly, any insulin in the culture medium may be susceptible to degradation and it has been shown by Ziegler et al. [11] that depending on the batches and concentrations of newborn calf serum used in the culture medium, the insulin recovery after 24-h culture at 37°C varied from 75 to 105%. Adding [3H]insulin to a culture system containing isolated islets, Halban et al. [12] have also found such extracellular degradation of insulin over time as up to 20% at the end of 6 days of culture. However, in the present study, the culture lasted for 3 days, and as insulin released towards the end of the culture period would be expected to be degraded to a lesser extent, and the insulin content of cells which had detached from the islets during the culture period was never found to contribute significantly to the total recoverable insulin of the culture system [12], no correction factor in augmenting the measured values for insulin accumulated in the culture medium has been taken into account. Furthermore, the anti-bovine insulin antibodies used in our study coupled with CNBr-activated Sepharose-4B have been found to bind insulin, proinsulin and even preproinsulin, and hence are denoted as (pro)insulin [13]. It has also been observed that in vitro formation of proinsulin in pancreatic islets is almost complete within the first 30 min of incubation and its conversion to insulin molecule in the fl-granules starts afterwards [14,15]. In the present study, as we have extended the culture period to 3 days, formation of labelled proinsulin and its conversion to insulin might have proceeded to a considerable extent. Therefore, a decrease in labelled (pro)insulin could not be the result solely of conversion of proinsulin to insulin without any degradation. Earlier studies [16,17] have also shown that glucose induces insignificant changes in specific radioactivity of the free intracellular leucine pool. Therefore, the methodology employed justified the practice of

considering the amount of radioactive amino acid incorporation into (pro)insulin as an index for quantification of (pro)insulin biosynthesis. However, despite the fact that such intra-insular degradation of insulin appeared to be regulated not only by the insulin release but by the glucose concentration per se [1,2], proteases of lysosomes [4] or microsomes [5,6] were thought to be involved in the process of insulin degradation. As chloroquine is readily taken up and selectively concentrated within the lysosomes (hence, lysosomotropic agent), and inhibits intralysosomal proteolysis [8,18-20], we tested whether addition of chloroquine could inhibit low-glucose mediated degradation of intra-insular insulin, but, as shown in Fig. 1, 0.02 mM cbloroquine failed to bring about any change in the low-glucose mediated degradation of insulin. On the other hand, as shown in Table 1, this concentration of chloroquine could significantly inhibit (pro)insulin biosynthesis in freshly isolated as well as precultured islets in the presence of either 2.2 or 22 mM glucose. In addition to that, chloroquine caused an insignificant increase in the release of immunoreactive insulin from the freshly isolated (at 22 mM glucose) as well as precultured islets (at 2.2 mM glucose) (Fig. 2, left lower and right upper panels). The failure,to inhibit low-glucose niediated degradation of intra-insular insulin by the concentration of chloroquine used in our study indirectly suggests that the intra-insular insulin degradation process takes place somewhere other than in the islet lysosomes. However, recently it has been immunocytochemically demonstrated that pancreatic islet lysosomes contain insulin and this has been put forward as evidence for islet lysosomal participation in the process of intracellular insulin degradation [7]. In different sets of experiments with cultured hepatocytes, however, chloroquine was found to have no significant effect on insulin processing at least at 37°C [21], and lysosomal degradation of insulin as a minor degradation pathway [22] and the probability of a non-lysosomal pathway of insulin degradation have been postulated. However, the quantitative role of lysosomal insulin degradation in overall insulin processing remains to be clar-

14 ified. In c o n t r a s t , a r e p o r t has been m a d e where in vivo a d m i n i s t r a t i o n o f c h l o r o q u i n e caused inhibition o f insulin d e g r a d a t i o n in a d i a b e t i c p a t i e n t with severe insulin resistance [23]. In a d d i t i o n , earlier studies have shown that there is a s t r o n g positive c o r r e l a t i o n between the activities o f islet l y s o s o m a l m a r k e r enzymes, e.g., acid p h o s p h a t a s e ( E C 3.1.3.2) a n d c a t h e p s i n - B (EC 3.4.22.1)-like enzymes a n d (pro)insulin b i o s y n t h e s i s in vitro [24-26]. Islet l y s o s o m a l enzymes have also been t h o u g h t to p a r t i c i p a t e in the c o n v e r s i o n mecha n i s m o f p r o i n s u l i n to insulin [26-28]. T h e r e f o r e , a l t h o u g h we have not here d e t e r m i n e d s e p a r a t e l y the i n d i v i d u a l biosynthesis o f p r o i n s u l i n a n d insulin, the a b o v e findings m i g h t s u p p o r t the h y p o t h e s i s that c h l o r o q u i n e - i n d u c e d interference o f islet lysos o m a l enzymes caused the in vitro i n h i b i t i o n o f (pro)insulin b i o s y n t h e s i s o b s e r v e d in o u r study. F r o m a survey o f the a v a i l a b l e literature on the use o f in vitro c o n c e n t r a t i o n s o f c h l o r o q u i n e [21,29], the possibility o f a toxic effect o f 0.02 m M c h l o r o quine causing interference with the (pro)insulin biosynthetic m a c h i n e r y in vitro does n o t seem to arise. C h l o r o q u i n e inactivates l y s o s o m a l p r o t e o l y t i c enzymes by increasing i n t r a l y s o s o m a l p H [19,20], a n d it has been r e p o r t e d that a l t e r a t i o n o f intracellular p H caused parallel effects on glucose-ind u c e d insulin secretory events [30]. H o w e v e r , studies by W o l l h e i m et al. [31] have f o u n d a lack o f c o r r e l a t i o n between n u t r i e n t - i n d u c e d c h a n g e s o f cytosolic p H a n d insulin release. Thus, it is now difficult to explain w h e t h e r the insignificant increase o b s e r v e d in the release o f i m m u n o r e a c t i v e insulin under the influence o f c h l o r o q u i n e is due to the c h l o r o q u i n e - i n d u c e d increase in i n t r a c e l l u l a r p H , or if this should be c o n s i d e r e d as a non-specific leakage p h e n o m e n o n o f insulin caused by a possible toxic action o f the c o n c e n t r a t i o n o f c h l o r o q u i n e employed. F r o m the results o f the present s t u d y it a p p e a r s that the p h y s i o l o g i c a l significance o f the l y s o s o m a l p a t h w a y o f i n t r a - i n s u l a r d e g r a d a t i o n o f insulin rem a i n s to be further elucidated. H o w e v e r , it is clear that a l t h o u g h c h l o r o q u i n e does not influence int r a - i n s u l a r d e g r a d a t i o n o f insulin, it does interfere with the biosynthesis o f insulin in the/,C-cells.

Acknowledgements T h a n k s are due to Mr. A. Ullrich for his technical assistance a n d to the A l e x a n d e r von H u m b o l d t F o u n d a t i o n for a w a r d i n g a fellowship to A . K . Chatterjee; this w o r k was s u p p o r t e d by the Deutsche Forschungsgemeinschaft, Project Scha/281.

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