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-glucoside hydrolase activities in parallel with glucose-stimulated insulin secretion
Department of Experimental Medical Science, University of Lund, BMC F13, S-221 84 Lund, Sweden
(Requests for offprints should be addressed to H Mosén; Email: henrik.mosen{at}med.lu.se)
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Abstract |
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-glucoside hydrolases, enzymes which we previously have shown to be implicated in the insulin release process. We have shown here that exogenous NO gas inhibits, while CO gas amplifies glucose-stimulated insulin secretion in intact mouse islets concomitant with a marked inhibition (NO) and a marked activation (CO) of the activities of the lysosomal/vacuolar enzymes acid glucan-1,4--glucosidase and acid -glucosidase (acid -glucoside hydrolases). Furthermore, CO dose-dependently potentiated glucose-stimulated insulin secretion in the range 0.1–1000 µM. In intact islets, the heme oxygenase substrate hemin markedly amplified glucose-stimulated insulin release, an effect which was accompanied by an increased activity of the acid -glucoside hydrolases. These effects were partially suppressed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Hemin also inhibited inducible NO synthase (iNOS)-derived NO production probably through a direct effect of CO on the NOS enzyme. Further, exogenous CO raised the content of both cGMP and cAMP in parallel with a marked amplification of glucose-stimulated insulin release, while exogenous NO suppressed insulin release and cAMP, leaving cGMP unaffected. Emiglitate, a selective inhibitor of -glucoside hydrolase activities, was able to markedly inhibit the stimulatory effect of exogenous CO on both glucose-stimulated insulin secretion and the activityof acid glucan-1,4--glucosidase and acid -glucosidase, while no appreciable effect on the activities of other lysosomal enzyme activities measured was found. We propose that CO and NO, both produced in significant quantities in the islets of Langerhans, have interacting regulatory roles on glucose-stimulated insulin secretion. This regulation is, at least in part, transduced through the activity of cGMP and the lysosomal/vacuolar system and the associated acid -glucoside hydrolases, but probably also through a direct effect on the cAMP system. |
Introduction |
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Carbon monoxide (CO), another small molecule, is formed from heme under the influence of the enzyme heme oxygenase (HO; Maines 1988, Marks 1994). There are two major known isoforms: an inducible enzyme (HO-1) and a constitutive enzyme (HO-2), both of which have been found in the islets of Langerhans (Henningsson et al. 1997, 1999, 2001, 2002, Ye & Laychock 1998, Alm et al. 1999, Lundquist et al. 2003, Mosén et al. 2005). Interestingly, addition of gaseous CO has an inhibitory effect on the production of NO in isolated islets (Henningsson et al. 2001) and very recently (Mosén et al. 2005), we found that an animal model of mild type 2 diabetes, the Goto-Kakizaki (GK) rat, showed a markedly reduced expression and activity of HO-2 in their islets. Moreover, in isolated islets, the cAMP system as well as CO antagonize the suppressive effect of NO on the insulin secretory process (Henningsson et al. 1999, 2000, Salehi et al. 2003, Jimenez-Feltstrom et al. 2004, 2005).
We have previously shown that one of the several secretory signals in nutrient-stimulated insulin release seems to be transduced by the activation of the glycogen hydrolyzing enzymes labelled acid -glucoside hydrolases, characterized as acid glucan-1,4--glucosidase and acid -glucosidase, both displaying a high activity in islet tissue (Lundquist 1985, 1986, Lundquist & Panagiotidis 1992, Salehi & Lundquist 1993a,1993b,1993c,1993d, Salehi et al. 1999, Mosén et al. 2000). The acid glucan-1,4--glucosidase preferentially cleaves -1,4-linked glucose polymers and produces free glucose, by attacking vacuolar glycogen, while the acid -glucosidase prefers oligosaccharides. However, their action and physiological effects are overlap considerably, (Pazur & Kleppe 1962, Smith et al. 1968, Lundquist 1985, 1986). The liberated glucose molecules could then serve as regulatory transducers, acting from the vacuolar compartment, in distal steps of the complex process of insulin release (Lundquist et al. 1996, Salehi et al. 1998b, 1999).
In a previous study (Mosén et al. 2000), we found that the intracellular NO donor hydroxylamine inhibited the acid -glucoside hydrolase activities in incubated intact mouse islets in parallel with a suppression of glucose-stimulated insulin release. In contrast, we have also observed (Henningsson et al. 1997, 1999, Mosén et al. 2005) that glucose stimulates islet production of CO in parallel with an enhanced insulin release.
Hence, the aim of this study was to focus on the action and interaction of NO and CO on glucose-stimulated insulin release, and to investigate whether the effects of these two gaseous messenger molecules might influence the insulin secretory pathway transduced through the lysosomal/vacuolar system and the activities of acid -glucoside hydrolases.
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Materials and Methods |
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Female mice of the naval medical research institute strain (B&K, Sollentuna, Sweden), weighing 25–30 g, standard pellet diet (B&K) and water available ad libitum, were used throughout the study. The animal experiments were approved by the local animal welfare committee (Lund, Sweden) and in accordance with the international standard recommended by NIH.
Drugs and chemicals
Collagenase (CLS 4) was obtained from Worthington Biochemicals (Freehold, NJ, USA), BSA and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) from ICN Biomedicals (High Wycombe, UK). Hemin, Rp-cAMPS and methylumbelliferyl-coupled substrates were from Sigma Chemical. N-[ß-(4-Ethoxycarbonylphenoxy)ethyl]-1-deoxy-nojirimycin (emiglitate) was kindly supplied by Bayer AG. All other chemicals were from British Drug Houses Ltd (Poole, UK) or Merck AG. The RIA kits for insulin determination were obtained from Diagnostica (Falkenberg, Sweden). Hemin was dissolved in 0.1 mol/l NaOH, followed by titration with 0.1 mol/l HCl.
Experimental procedure
Isolation of pancreatic islets from freely fed mice was accomplished by retrograde injection of a collagenase solution via the bile-pancreatic duct (Gotoh et al. 1985).
Experiments with intact islets
Freshly isolated islets were preincubated for 30 min at 37 °C in Krebs Ringer bicarbonate buffer (pH 7.4), supplemented with 10 mM HEPES, 0.1% BSA and 1 mM glucose. When only insulin secretion was studied, we used batches of ten islets in 1 ml incubation medium. When islet lysosomal enzymes were determined as well, batches of 30 islets in 1.5 ml were used. In the experiments where emiglitate was used, it was present in the preincubation medium as well. After preincubation at 1 mM glucose for 30 min, the buffer was changed to a medium containing 1, 12 or 20 mM glucose, as well as the different test agents used. The islets were incubated in a water bath at 37 °C (30 cycles/min). In the experiments where exogenous NO or CO was used, the incubation buffer was purged of O2 by helium and saturated with either NO or CO and the control buffer was saturated with helium. The solubility of CO when saturated is 2.3 ml/100 ml H2O (
1 mM) and of NO 4.6 ml/100 ml H2O ( 2 mM). To achieve CO concentrations ranging from 0 to 1000 µM, the incubation solution was diluted with an identical buffer solution saturated with CO. In the experiments where NOS activities were studied, batches of 200 islets in 1.5 ml incubation medium were used.
Immediately after incubation, an aliquot of the medium was removed for an assay of insulin in all the experiments with intact islets. In the experiments where islet lysosomal enzyme activities were measured, the islets were then thoroughly washed and collected in 200 µl ice-cold acetate–EDTA buffer (1.1 mM EDTA and 5 mM Na acetate (pH 5.0)) and thereafter stored at –20 °C. In the experiments where NOS activities were determined, the islets were instead collected in 840 µl ice-cold buffer, containing 20 mmol/l N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.5 mmol/l EDTA, and 1 mmol/l D,L-dithiothreitol (pH 7.2). On the day of assay, the islets were sonicated on ice and enzyme activities were determined.
HPLC analysis of islet NO production When NOS activities were measured the buffer solution containing the islet homogenate was reconstituted to contain, in addition to the above-mentioned compounds, also 0.2 mmol/l L-arginine, 0.45 mmol/l CaCl2, 2 mmol/l NADPH and 25 U calmodulin in a total volume of 1 ml. To determine iNOS activity, both Ca2+ and calmodulin were omitted. The buffer composition was essentially the same as previously described for the assay of NOS in brain tissue using radiolabelled L-arginine (Bredt & Snyder 1989). Aliquots of the incubated homogenate (200 µl) were then passed through a 1 ml Amprep chloro benzoic acid cation-exchange column for HPLC analysis of the L-citrulline formed according to Carlberg and others (Carlberg 1994, Salehi et al. 1996, Henningsson et al. 2002). Since L-citrulline is created in equimolar concentrations to NO and since L-citrulline is stable, whereas NO is not, L-citrulline is the preferred parameter when measuring NO production.
Assay of islet lysosomal enzyme activities
The procedures for determination of acid phosphatase (pH 4.5), acid -glucosidase (pH 4.0) and N-acetyl-ß-D-glucosaminidase (pH 5.0) with methylumbelliferyl-coupled substrates have previously been described (Lundquist 1985). Islet glucan-1,4--glucosidase activity with glycogen as substrate was determined at pH 4.0 as described in detail elsewhere (Lundquist 1971, 1985). Protein was analysed according to the method of Lowry et al.(1951).
Determination of cAMP and cGMP Incubation of isolated islets was performed in the presence of phosphodi-esterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mmol/l) and was stopped by removal of the buffer and the addition of 0.5 ml ice-cold 10% trichloroacetic acid (TCA), followed by immediate freezing in a –70 °C ethanol bath (Panagiotidis et al. 1995). Before the assay, 0.5 ml H2O was added, and the samples were sonicated for 3 x 5 s followed by centrifugation at 1100 g for 15 min. The supernatants were collected and extracted with 4 x 2 ml water-saturated diethyl ether. The aqueous phase was removed and freeze-dried, using a Lyovac GT 2 freeze dryer. The residue was then dissolved in 450 µl of 50 mmol/l Na-acetate buffer (pH 6.2). The amounts of cAMP and cGMP were quantified with a [125I]-cAMP and [125I]-cGMP RIA kit (RIANEN, Du Pont Company, Boston, MA, USA). [3H]-cGMP was added to the TCA islet homogenate in order to determine the recovery of cAMP and cGMP during the ether extraction. The mean recovery was 85%.
Determination of insulin Insulin was determined with a RIA (Heding 1966).
Statistical analysis
Statistical significance between the sets of data was assessed using unpaired Student’s t-test with Welch correction when appropriate, or where applicable, ANOVA followed by Tukey’s multiple comparisons test. Results are expressed as means ± S.E.M.
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Results |
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Table 1
shows the effect of NO and CO gas on lysosomal enzyme activities in islet homogenates. Addition of NO or CO gas in aqueous solution (saturating the homogenate buffer) induced quite different effects on the enzyme activities. NO suppressed the acid -glucoside hydrolase activities by approximately 60% and the activities of acid phosphatase and N-acetyl-ß-D-glucosaminidase by 30%. In contrast, CO greatly stimulated all enzyme activities by ~150–250% (Table 1).
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Figures 1 and 2 show the effects on insulin release and lysosomal enzyme activities after the incubation of intact islets at a glucose concentration of 1 or 20 mM, in buffer solutions saturated with either NO ( 2 mM) or CO ( 1 mM). Figure 1 shows the effect of NO, which displayed an inhibitory action on both basal and glucose-stimulated insulin secretion. The effect was quite impressive at high glucose concentration where the insulin secretion was almost abrogated to a level not different from the basal levels. Concomitant with this effect, a similar suppressive action on the activities of acid glucan-1,4--glucosidase as well as its isoform acid -glucosidase was seen, while the activities of acid phosphatase and N-acetyl-ß-D-glucosaminidase were unaffected.
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shows that, in contrast to NO, addition of CO to the incubation medium had a pronounced stimulatory effect not only on the acid -glucoside hydrolases, but also on the other lysosomal enzymes, both at low and high glucose concentration. The stimulatory effect by CO on the lysosomal enzyme activities at high glucose concentration was associated with a parallel amplification of glucose-induced insulin release, while basal insulin release at low glucose concentration was not affected (Fig. 2). Effect of the HO substrate hemin on glucose-stimulated insulin release and islet lysosomal enzyme activities
We have previously shown that glucose stimulates islet CO production concomitant with an amplification of insulin release (Henningsson et al. 1997, 1999, Mosén et al. 2005). Figure 3 shows that the HO substrate hemin amplified glucose-induced insulin release in parallel with a marked stimulation of islet lysosomal enzyme activities. This hemin-stimulated increase in insulin release and lysosomal enzyme activities was greatly reduced by ODQ, a selective inhibitor of soluble guanylate cyclase (Fig. 3). In the absence of hemin, ODQ slightly suppressed glucose-stimulated insulin release as well as acid -glucosidase and acid phosphatase activities (Fig. 3).
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To further investigate the influence of the HO–CO signalling pathway on glucose-stimulated insulin release, we performed a dose–response study with increasing concentrations of exogenously added CO gas. Figure 4 shows that CO amplifies insulin release also at a modestly ‘hyperglycaemic’ concentration of glucose (12 mM). This effect was barely detectable at 0$1 mM CO and showed a broad maximum at 10–1000 µM. A similar dose–response range for such an amplification by CO on glucose-stimulated insulin release was noted at 20 mM glucose (data not shown).
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Isolated islets were incubated at 12 mM glucose and 100 µM CO in the absence and presence of the PKA-inhibitor Rp-cAMPS, the PKC-inhibitor bisindolylmaleimide and the guanylate cyclase-inhibitor ODQ. Figure 5 shows that the CO-stimulated amplification of glucose-stimulated insulin release was significantly reduced by the PKA and the guanylate cyclase inhibitors, while the PKC inhibitor did not influence the CO-induced effect.
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The results shown in Fig. 5 suggested that both the cAMP and the cGMP pathways are implicated in the CO-induced amplification of glucose-stimulated insulin release. Because NO as well as CO are known to stimulate the cGMP system in various cell types (Moncada et al. 1991), we conducted a series of experiments measuring the cyclic nucleotide content of isolated islets after incubation at high glucose concentration (20 mM) in the absence and presence of CO or NO gas (saturated concentrations). Figure 6 shows that CO raised the content of both cGMP and cAMP in parallel with a marked amplification of glucose-stimulated insulin release. In contrast, NO greatly suppressed insulin release and the cAMP content, while the cGMP content was not significantly affected. Figure 6 also shows the islet content of both nucleotides and that, as expected, insulin release was raised by glucose itself when increasing the glucose concentration from 1 to 20 mM.
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The interaction of CO and NO on glucose-stimulated insulin release was tested by studying the influence of hemin on the activities of islet isoforms of NOS. Figure 7 shows that the increase in iNOS activity after incubation of isolated islets at 20 mM glucose was greatly reduced by the addition of hemin to the incubation medium. This inhibition of iNOS activity by hemin was accompanied by an impressive amplification of glucose-induced insulin release. Hemin also tended to reduce glucose-stimulated ncNOS activity resulting in a highly significant inhibition of total NO production (Fig. 7).
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-glucoside hydrolase inhibitor emiglitate on glucose-stimulated insulin release and islet lysosomal enzyme activities in the absence and presence of CO
To study further whether the downstream potentiating action of CO in glucose-stimulated insulin secretion would interact with the vacuolar system and the lysosomal enzyme activities, we performed a series of experiments with the selective -glucoside hydrolase inhibitor emiglitate. Figure 8 shows that, in control islets, emiglitate greatly suppressed the glucose-stimulated insulin release in parallel with an inhibitory effect on the activities of acid glucan-1,4--glucosidase and acid -glucosidase. In contrast, the activities of acid phosphatase and N-acetyl-ß-D-glucosaminidase tended to increase in the presence of the -glucoside hydrolase inhibitor. Moreover, the CO-induced amplification of the glucose-stimulated insulin release as well as of the increased activities of the acid -glucoside hydrolases were abrogated by emiglitate and displayed the same levels as in the absence of CO (Fig. 8). The CO-induced rise in the activities of acid phosphatase and acid N-acetyl-ß-D-glucosaminidase was not appreciably affected by emiglitate (Fig. 8).
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Discussion |
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-glucoside hydrolases in intact incubated islets concomitant with a marked inhibition of glucose-stimulated insulin release. This is in accordance with our previous results showing that suppression of islet acid -glucoside hydrolase activities by various means is accompanied by a similar parallel inhibition of glucose-stimulated insulin release (Salehi & Lundquist 1993a,1993b,1993c, 1996, Salehi et al. 1995, 1998b, 1999, Lundquist et al. 1996, Mosén et al. 2000), suggesting an intact activity of these enzymes being one of the several important links in the insulin secretory process induced by glucose. With regard to the inhibitory effect of NO in this context, it seems most likely that important thiol groups on the enzyme molecule are nitrosylated and thus inactivated (Jaffrey et al. 2001), because the acid -glucoside hydrolases are known to contain several cysteine residues (Barrett 1972) and it has been shown that inactivation of acid -glucosidase from cattle liver can be prevented by protecting the sulphydry-groups (Bruni et al. 1969).
Stimulation of the islet acid -glucoside hydrolase transduction system, e.g. by Ca2+ and/or nutrient secretagogues leads to an increased insulin release (Salehi & Lundquist 1993b,c, Lundquist et al. 1996, Salehi et al. 1998b, Mosén et al. 2000). However, apart from Ca2+, no established intracellular messenger molecules associated with activation of the acid -glucoside hydrolases and the lysosomal/vacuolar insulin secretory pathway are known. The present results suggest that the recently discovered HO–CO system (Henningsson et al. 1997, 1999, Lundquist et al. 2003, Mosén et al. 2005) might be a likely candidate in this regard. We show here that CO stimulates the acid -glucoside hydrolases in islet homogenates and, most important, also in intact islets concomitant with a strong increase in glucose-stimulated insulin release. Since we have previously shown that glucose itself induces the production of CO in isolated islets (Henningsson et al. 1997, 1999, Mosén et al. 2005), it is tempting to suggest that glucose and/or its metabolism stimulates the production of CO, which in turn activates the acid -glucoside hydrolases eliciting a vacuolar compartmentalized signal, resulting in free glucose and/or modification of -1,4-linked glucose residues in membrane glycoproteins taking part in the complex process of exocytosis. Interestingly, in addition, CO also has stimulatory effects on the activities of acid phosphatase and N-acetyl-ß-D-glucosaminidase when added to intact islets. This might indicate that CO has a general stimulatory or modifying effect on the whole acidic vacuolar compartment of the ß-cell, i.e. secretory granules as well as other lysosomal/vacuolar organelles. Such an explanation would be in accordance with our previous data from the mildly diabetic GK rat, an animal model of type 2 non-obese diabetes, showing that the defective insulin response to glucose might involve an impaired interaction between different organelle constituents within the islet vacuolar system (Salehi et al. 1999) and that this impairment is associated with a greatly reduced CO production in these islets (Mosén et al. 2005).
We have shown previously that the pancreatic islets display an unusually high activity of HO-2 and that the HO-2 protein is expressed in all four major types of islet endocrine cells (Henningsson et al. 1997, 1999, 2000, Alm et al. 1999, Lundquist et al. 2003, Mosén et al. 2005). Moreover, hemin was found to stimulate, while the HO inhibitor Zn-protoporphyrin suppressed glucose-induced insulin release in parallel with the rate of CO production (Henningsson et al. 1997, 1999, Lundquist et al. 2003). Further, the enhancement of glucose-stimulated insulin release induced by hemin could be markedly reduced by the selective guanylate cyclase inhibitor ODQ (Henningsson et al. 1999) suggesting that a major effect of CO on insulin release might be exerted through the guanylate cyclase–cGMP system. The present data in addition showed that the hemin-induced amplification of the insulin response to glucose was associated with increased activities of the acid -glucoside hydrolases as well as of acid phosphatase and N-acetyl-ß-D-glucosaminidase. The hemin-induced amplification of glucose-stimulated insulin release and the parallel increase in lysosomal enzyme activities were fully inhibited by ODQ, suggesting that the HO–CO–cGMP system not only amplifies glucose-stimulated insulin release but also has a general stimulating effect on the vacuolar system/lysosomal enzyme activities. However, ODQ added to the incubation medium at high glucose, but in the absence of hemin had only a slight inhibitory effect on glucose-stimulated insulin release and only marginally inhibited some of the lysosomal enzymes. These results thus raised the question whether the HO–CO signalling pathway might affect transduction mechanisms in glucose-stimulated insulin release other than the guanylate cyclase–cGMP system. CO dose-dependently amplified glucose-stimulated insulin release, and the results obtained in the presence of different antagonists against the cAMP (Rp-cAMPS), phospholipase C (bisindolylmaleimide) and cGMP (ODQ) systems, suggested that both the cGMP and the cAMP pathways were involved, while a direct effect on the phospholipase C pathway appeared less likely. The interaction of the HO–CO signalling pathway with the cAMP system was further corroborated by our observation that addition of gaseous CO to islets incubated at high glucose concentration amplified the glucose-stimulated increase in islet cAMP content in parallel with a similar enhancement of insulin release and a still more pronounced increase in islet cGMP content. In contrast, addition of gaseous NO to the incubation medium greatly suppressed the insulin release as well as the islet cAMP content, while the cGMP content was unaffected. These data suggested that the islet cAMP system is highly involved in the HO–CO signalling pathway and that CO, at least at high glucose concentration, has a greater impact than NO on the cGMP system, which at first seems to be at variance with the data from most other tissues (Ingi et al. 1996, Hartsfield 2002). However, it should be noted that endogenous molecules of an indazole nature have been hypothesized to sensitize the soluble guanylate cyclase to CO action (Hartsfield 2002). Although the interaction of NO and CO on the different NO- and CO-synthesizing enzymes is unclear and presently not predictable (Hartsfield 2002), we have found, in isolated islets, that iNOS-derived NO is inclined to stimulate HO-2-derived CO production in the absence of inflammatory processes (Henningsson et al. 2000), while an increased CO production emanating from both HO-1 and HO-2 is associated with iNOS-derived NO production after injection of endotoxin (Henningsson et al. 2001).
Hence, a high HO-derived CO production with the associated formation of the antioxidant bilirubin is probably beneficial as a protective mechanism against the deleterious action of iNOS-derived NO (Eizirik et al. 1996, Henningsson et al. 2001). Moreover, we have previously shown that addition of gaseous CO to islets incubated at high glucose greatly suppressed islet NO production, an effect which was not afflicted by ODQ and thus appeared to be operating independently of the cGMP system (Henningsson et al. 2001). This is consistent with the present data, which show that the inhibition of islet NO production by hemin was mainly exerted on iNOS-derived NO. In this context, it should be emphasized that islets freshly isolated ‘ex vivo’ from both mice and rats display a more than five- to tenfold higher production of CO than NO (Henningsson et al. 1997, 1999, 2000, 2001, 2002, Mosén et al. 2005), a difference that might partly compensate for the low levels of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase in the ß-cell (Mysore et al. 2005).
The present results suggest that the CO-induced amplification of glucose-stimulated insulin release is elicited both through the cGMP and the cAMP pathways, and that an important part of the cGMP effect is transduced through the activation of the acid -glucoside hydrolases and the lysosomal/vacuolar system, although an additional direct action of CO on this system cannot be excluded. In contrast to the involvement of the acid -glucoside hydrolases in insulin-releasing processes stimulated by glucose, the secretory pathways induced through direct activation of the cAMP or phospholipase C systems seem to operate independently of these enzyme activities (Salehi & Lundquist 1993a,1993b,1993c, Lundquist et al. 1996, Salehi et al. 1998b). In fact, suppression of glucose-stimulated insulin release by the selective -glucoside hydrolase inhibitor emiglitate, as well as a dysfunction in the interaction between acidic organelles, could be compensated for by stimulation of the cAMP pathway through the adenylate cyclase activator forskolin (Salehi & Lundquist 1996, Salehi et al. 1999).
The relative importance of the cGMP-acid -glucoside hydrolase pathway in relation to the cAMP pathway in CO-stimulated amplification of glucose-stimulated insulin release was shown by the fact that both ODQ and emiglitate suppressed a major part of this amplification, which suggests that cGMP is an important messenger in this context. However, as shown by the present results as well as by previous data (Lundquist et al. 1996, Henningsson et al. 1999) emiglitate greatly reduced the insulin response to glucose in the absence of exogenously added CO, while ODQ had only a minor inhibiting effect. Hence, most likely emiglitate inhibited not only the cGMP signalling pathway, but also the other transduction pathways in glucose-stimulated insulin release. The unique importance of an intact activity of the acid -glucoside hydrolases for the insulin response to glucose is emphasized by our previous data showing a pronounced inhibition of glucose-stimulated insulin release both in vitro and in vivo in the presence of selective -glucoside hydrolase inhibitors of different chemical nature, such as the deoxynojirimycin derivatives miglitol and emiglitate, the indolizine alkaloid castanospermine and the pseudotetrasaccharide acarbose (Salehi & Lundquist 1993b,c, Salehi et al. 1995, 1998b,, Salehi et al. c, 1999, Lundquist et al. 1996). The notion that acarbose accumulates specifically in the lysosomal/vacuolar system (Salehi et al. 1995, 1999) strengthens the validity of these data. It should be recalled that emiglitate and other selective -glucoside hydrolase inhibitors exert their inhibitory effect on glucose-induced insulin release at a distal step in the stimulus-secretion coupling, since they also inhibit insulin release stimulated by nutrients directly entering the mitochondrial metabolism, e.g. leucine and -ketoisocaproic acid, (Lundquist et al. 1996) and they do not influence glucose oxidation (Salehi et al. 1995). In contrast, a possible defect in endogenously produced CO is most likely associated with an earlier step in glucose-induced insulin release, since the glucose-stimulated induction of the HO–CO signalling pathway is defective in the islets of the diabetic GK rat, and these islets respond to exogenous CO with a normal amplification of glucose-stimulated insulin release (Mosén et al. 2005).
The present data show that two evolutionary very old messenger molecules, CO and NO, have a profound regulatory influence on glucose-stimulated insulin release and that the vacuole-associated glycogenolytic acid -glucoside hydrolases seem to be deeply involved in the action of these gaseous molecules. We have previously proposed that the action of the acid -glucoside hydrolases should take place in an acidic milieu at a distal step in the secretory process (Lundquist et al. 1996, Salehi et al. 1999) and probably in association with exocytosis at the entry of Ca2+ in the region of the ß-cell with the highest density of secretory granules (Bokvist et al. 1995). Such a hypothesis is in accordance with the observation in yeast (Ungermann et al. 1999) that vacuole acidification is required for the pairing of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which take part in the exocytotic process, and that granular acidification is involved in ß-cell exocytosis (Barg et al. 2001). Moreover, this hypothesis is further encouraged by a very recent finding (Yamasaki et al. 2004) that different secretory stimuli use different Ca2+ organelles to elicit unique responses. Thus, in the ß-cell, glucose was found to mobilize Ca2+ from a lysosome-related organelle, while cholinergic stimulation, having no effect on the acid -glucoside hydrolases (Salehi & Lundquist 1993a), only used a pathway associated with the endoplasmic reticulum (Yamasaki et al. 2004). Hence, we propose that further studies are required to resolve hitherto overlooked relationships between secretory granules and other acidic organelles in glucose-stimulated exocytosis and the involvement of CO and NO in these processes. It should be noted that it cannot be excluded that a fraction of the acid -glucoside hydrolases could be located within the secretory granules themselves. Figure 9 shows an over-simplified scheme on the proposed interaction of NO, CO and the acid -glucoside hydrolases in glucose-stimulated insulin release.
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Acknowledgements |
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References |
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P < 0.001.
