Diabetogenic effect of cyclosporin A is mediated by interference with mitochondrial function of pancreatic B-cells.

Treatment of patients after organ transplantation with the immunosuppressive drug cyclosporin A (CsA) is often accompanied by impaired glucose tolerance, thus promoting the development of diabetes mellitus. In the present article we show that 2 to 5 microM CsA diminishes glucose-induced insulin secretion of isolated mouse pancreatic islets in vitro by inhibiting glucose-stimulated oscillations of the cytoplasmic free-Ca(2+) concentration [Ca(2+)](c). This effect is not due to an inhibition of calcineurin, which mediates the immunosuppressive effect of CsA, because other calcineurin inhibitors, deltamethrin and tacrolimus, did not affect the oscillations in [Ca(2+)](c) of the B-cells. The CsA-induced decrease in [Ca(2+)](c) to basal values was not caused by a direct inhibition of L-type Ca(2+) channels. CsA is known to be a potent inhibitor of the mitochondrial permeability transition pore (PTP), which we recently suggested to be involved in the regulation of oscillations. Consequently, CsA also inhibited the oscillations of the cell membrane potential, and it is shown that these effects could not be ascribed to cellular ATP depletion. However, the mitochondrial membrane potential Delta Psi was affected by CsA by inhibiting the oscillations in Delta Psi. Interestingly, the observed reduction in [Ca(2+)](c) could be counteracted by the K(+)(ATP) channel blocker tolbutamide, indicating that the stimulus-secretion coupling was interrupted before the closure of K(+)(ATP) channels. It is concluded that CsA alters B-cell function by inhibiting the mitochondrial PTP. This terminates the oscillatory activity that is indispensable for adequate insulin secretion. Thus, CsA acts on different targets to induce the immunosuppressive and the diabetogenic effect.

Modulation of beta-cell ouabain-sensitive 86Rb+ influx (Na+/K+ pump) by D-glucose, glibenclamide or diazoxide.

The activity of the beta-cell Na+/K+ pump was studied by using ouabain-sensitive (1mM ouabain) 86Rb+ influx in beta-cell-rich islets of Umeå-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive 86Rb+ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na+/K+ pump. Pre-treatment (60 or 180 min) of islets at 0 mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of 86Rb+ influx during the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive 86Rb+ uptake in the same manner as glucose. The stimulatory effect showed its apparent maximum at 0.5 microM. Pre-treatment (60 min) of islets with 1 microM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive 86Rb+ influx. The stimulatory effect of glibenclamide and D-glucose were not additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1 microM) was observed on the Na+/K+ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na+/K+ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na+/K+ pump, probably as part of the membrane repolarisation process.

Methyl pyruvate initiates membrane depolarization and insulin release by metabolic factors other than ATP.

The role of mitochondria in stimulus-secretion coupling of pancreatic beta-cells was examined using methyl pyruvate (MP). MP stimulated insulin secretion in the absence of glucose, with maximal effect at 5 mM. K+ (30 mM) alone, or in combination with diazoxide (100 microM), failed to enhance MP-induced secretion. Diazoxide (100 microM) inhibited MP-induced insulin secretion. MP depolarized the beta-cell in a concentration-dependent manner (5-20 mM). The sustained depolarization induced by 20 mM MP was not influenced by 100 microM diazoxide, but the continuous spiking activity was suppressed by 500 microM diazoxide. Pyruvate failed to initiate insulin release (5-20 mM) or to depolarize the membrane potential. ATP production in isolated beta-cell mitochondria was detected as accumulation of ATP in the medium during incubation in the presence of malate or glutamate in combination with pyruvate or MP. There was no difference in ATP production induced by pyruvate/malate or MP/malate in isolated beta-cell mitochondria. ATP production by MP/glutamate was higher than that induced by pyruvate/glutamate, but it was much lower than that induced by alpha-ketoisocaproate/glutamate. Pyruvate (5 mM) or MP (5 mM) had no effect on the ATP/ADP ratio in whole islets, whereas glucose (20 mM) significantly increased the whole islet ATP/ADP ratio. It is concluded that MP-induced beta-cell membrane depolarization or insulin release does not relate directly to mitochondrial ATP production. Instead MP may exert a direct extramitochondrial effect, or it may stimulate beta-cell mitochondria to produce coupling factors different from ATP to initiate insulin release.

Relationships between the Na(+)/K(+) pump and ATP and ADP content in mouse pancreatic islets: effects of meglitinide and glibenclamide.

We have previously demonstrated that both D-glucose and glibenclamide stimulate the Na(+)/K(+) pump and suggested that this may be part of the membrane repolarization process, following the primary depolarization by these agents. The aim of this study was to investigate whether the non-sulphonylurea meglitinide (HB 699) exerts similar effects as glibenclamide or glucose on the islet Na(+)/K(+) pump and if effects of meglitinide or glibenclamide on this pump activity is paralleled by changes in islet ATP content and/or ATP/ADP ratio. The acyl-amino-alkyl benzoic acid derivative, meglitinide, stimulated the islet ouabain-sensitive portion of (86)Rb(+) influx (Na(+)/K(+) pump) by 53%, while the ouabain-resistant portion was inhibited by 70%. The stimulatory effect was not additive to that caused by D-glucose, suggesting that both agents may activate the Na(+)/K(+) pump via the same mechanism. Glibenclamide (10 microM) decreased the islet ATP and ADP content as well as the ATP/ADP ratio at 0 mM glucose. These effects were no longer observed at 10 mM glucose. Meglitinide (10 or 50 microM) lowered the islet ATP and ADP content at 0 mM glucose without affecting the ATP/ADP ratio. At 10 mM glucose, however, 10 microM of the drug reduced the islet ATP content but not the ATP/ADP ratio, while 50 microM of the drug, besides lowering the ATP content, also reduced the ATP/ADP ratio. Diazoxide (0.5 mM) increased the islet ATP content in the absence of glucose, an effect not seen in the presence of 10 mM glucose. The rate of glucose oxidation at 1, 10 or 20 mM of the sugar was not affected by glibenclamide (0.1 - 10 microM) and at 10 or 20 mM of the sugar not affected by meglitinide (1 - 100 microM). These results suggest that glibenclamide and meglitinide lower the islet ATP level by indirectly activating the beta-cell Na(+)/K(+) pump, which is a major consumer of ATP in the islets, while diazoxide increases the ATP level due to inhibition of the pump.

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells.

To determine the effect of D-glucose on the beta-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Umeå-ob/ob mice in the absence or presence of 1mM ouabain. D-glucose (20mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Umeå-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3-20mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135mM Na+ and 20mM K+). Neither D-glucose (3-20mM) nor L-glucose or 3-O-methyl-D-glucose (20mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7 mM K+). Diphenylhydantoin (150 microM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 microM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of beta-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.

Lactate production in pancreatic islets.

Lactate production, glucose utilization, glucose oxidation, and insulin release were studied in islets from rat and ob/ob mice. Lactate was determined with a highly sensitive method, based on esterification, subsequent separation, and quantitation with high-performance liquid chromatography. There was a significant lactate production in the absence of glucose, which increased with glucose concentrations up to 3 mmol/l, reaching its half-maximal rate in the presence of 0.2-1.0 mmol/l glucose in both species. Glucose utilization displayed a wider glucose concentration dependence, with a K0.5 value between 3 and 10 mmol/l glucose. The rates of glucose utilization and lactate production were similar at 3 mmol/l glucose in rat islets and at about 6 mmol/l glucose in ob/ob mice islets. Saturation of lactate production at low glucose concentrations is probably contributing to the observed preferential stimulation of oxidative metabolism at higher concentrations. D-Mannoheptulose caused a marked inhibition of glucose utilization and glucose oxidation at 20 mmol/l glucose in islets from rat or ob/ob mice, as would be expected from a competitive inhibition of glucokinase. By contrast, D-mannoheptulose reduced only marginally the islet metabolism at 3 mmol/l glucose, which is consistent with an effective mannoheptulose-induced inhibition of the glucokinase-dependent, minor part of glucose phosphorylation at this low glucose concentration.

Alpha-ketoisocaproate is not a true substrate for ATP production by pancreatic beta-cell mitochondria.

The ability of alpha-ketoisocaproate (KIC) to induce ATP production in isolated mitochondria from pancreatic beta-cells was examined with a bioluminometric method. There was no ATP production from KIC when tested alone or in combination with malate (1 mmol/l), nor did DL-beta-hydroxybutyrate induce mitochondrial ATP production, whereas palmitoyl-carnitine and pyruvate were efficient stimulators of mitochondrial ATP production in the presence of an equimolar concentration of malate. However, KIC stimulated the mitochondrial ATP production when tested in combination with glutamate (10 mmol/l). The concentration necessary to obtain half-maximal stimulation was approximately 50 micromol/l KIC, and maximal activity, comparable to that obtained with fatty acids, was reached at 1 mmol/l KIC. Higher KIC concentrations inhibited the mitochondrial ATP production, whereas a plateau was attained at 1 mmol/l KIC in the presence of glutamine. Ca2+ stimulated the maximal mitochondrial ATP production induced by KIC. Maximal stimulation was obtained with 300 nmol/l Ca2+ in the presence of 0.3 mmol/l KIC. Ca2+ reduced the concentration of KIC necessary for half-maximal stimulation to <30 micromol/l. Leucine stimulated the mitochondrial ATP production in the presence of glutamate to the same extent as KIC. Half-maximal stimulation was observed with 2 mmol/l leucine. There were no additive effects on mitochondrial ATP production when KIC and leucine were tested in combination. The results demonstrate that KIC by itself is not a mitochondrial substrate for ATP production. KIC must transaminate with glutamate or glutamine to yield alpha-ketoglutarate and leucine. Since leucine allosterically activates glutamate dehydrogenase, which also produces alpha-ketoglutarate, the insulinogenic effect of KIC may in part be due to the intramitochondrial generation of alpha-ketoglutarate. Since KIC-induced ATP production reaches a plateau already at micromolar concentrations (i.e., far below the concentrations at which KIC induces insulin release), it is proposed here that the catabolism of KIC may induce additional signals related to insulin release.

Glycerol 3-phosphate-induced ATP production in intact mitochondria from pancreatic B-cells.

A bioluminescent method is presented that allows monitoring of ATP production from mitochondria corresponding to one islet of Langerhans per sample. In mitochondria from ob/ob mice Ca2+ stimulates the ATP production in the presence of L-glycerol 3-phosphate (GP) by reducing the Km for GP by one order of magnitude to about 3 mM. Maximal ATP production in the presence of Ca2+ (200 nM) is obtained at 10 mM GP. The free calcium concentration required to reach half-maximal stimulation (K0.5Ca2+) depends on the GP concentration, thus half-maximal effects are observed at about 80 nM at low GP (1 mM) and 10 nM at high GP (10 mM). Sodium can replace Ca2+ as a stimulator of GP-induced ATP production. It activates ADP phosphorylation by B-cell mitochondria in a sigmoidal concentration-dependent manner in the absence of Cs2+ (Hill coefficient 2.3 +/- 0.2) but does not change K0.5ca2+ nor the maximal mitochondrial activity. Ca2+ concentrations higher than 300 nM are inhibitory at all tested substrate concentrations. Mitochondria from ob/ob mice showed no functional defect when compared with normal controls. It is concluded that activation of the glycerol phosphate shuttle may not be the main coupling site for glucose-induced insulin release at maximal cytoplasmic Ca2+ levels.

Regulatory effects of ATP and luciferin on firefly luciferase activity.

ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. High concentrations of albumin reduce this luciferin effect. The kinetic properties of firefly luciferase determined from initial rates and at steady state after 1 min of catalysis have been analysed according to Michaelis-Menten kinetics. There is only one active site for each of the substrates. At steady state the Km and Vmax. values for both substrates are reduced in an uncompetitive manner. Hyperbolic Lineweaver-Burk plots indicate an activation by ATP probably by binding to an allosteric site. A model is presented which incorporates luciferin induced de- and re-activation effects. Experimental conditions to avoid the regulatory effects of substrates during ATP monitoring are proposed.

Insulin, glucagon, somatostatin, and thyrotropin-releasing hormone content and secretion by perifused fetal rat islets during culture.

In the neonatal period of the rat, pancreatic thyrotropin-releasing hormone content decreases and the sensitivity of insulin secretion to glucose increases. In adult rat islets, TRH inhibits glucose-induced insulin release. The aim of this study was to investigate whether a high TRH content and release can be part of the explanation for the functional immaturity of neonatal islets. For that purpose, we have measured the tissue content and the secretion of immunoreactive insulin, glucagon, somatostatin and TRH in islets from 21.5-day-old rat fetuses cultured for up to one week. Insulin, glucagon and somatostatin content increased during one week of culture in the presence of 11.1 mmol/l glucose. The TRH content decreased during culture, but did not equal adult values. Insulin, glucagon and somatostatin responses to glucose were present after one week of culture. Glucose had no effect on TRH release in cultured fetal islets, but inhibited TRH release in adult islets. We conclude that glucose can stimulate insulin secretion without inhibiting TRH release, but that a decrease in islet TRH content and a sensitization of TRH secretion to glucose may be important in the full maturation of fetal pancreatic islets.

Middle ear effusion induced by various inflammatory mediators and neuropeptides. An experimental study in the rat.

Vascular leakage in the middle ear cavity was studied after i.v. administration of various substances in rats and determined by the Evans blue technique. Bradykinin, histamine, serotonin, acetylcholine, substance P (SP) and vasoactive intestinal polypeptide (VIP) resulted in extravasation of Evans blue. In the case of bradykinin and histamine, the leakage was dose dependent. Calcitonin gene-related peptide (CGRP) did not affect vessel permeability. In other experiments the effect of histamine antagonists was tested on production of middle ear effusion, caused by blowing air at 14 degrees C into the external auditory canal (EAC). The increase in vessel permeability in this otitis media model was inhibited by the H2-receptor antagonist cimetidine, at doses 0.1 and 1.0 mg/ml. Diphenhydramine, an H1-receptor antagonist, arrested only partly middle ear fluid accumulation. Our study demonstrated that various inflammatory mediators and neuropeptides are capable of inducing vascular leakage in the middle ear cavity. It was also concluded that H2-receptors are involved in the regulation of middle ear vascular permeability.

Measurements of serum glucose using the luciferin/luciferase system and a liquid scintillation spectrometer.

A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.

Effects of alpha-adrenoceptor agonists and antagonists on insulin secretion, calcium uptake, and rubidium efflux in mouse pancreatic islets.

Epinephrine, norepinephrine or the more selective alpha-2 adrenoceptor agonist, clonidine, inhibited insulin release from isolated pancreatic islets of lean mice or obese mice homozygous for the gene ob. Clonidine was highly effective at 0.1 mumol/l. In contrast, the preferential alpha-1 adrenoceptor agonist, phenylephrine, had no or only a modest effect at 10 mumol/l. The effects of norepinephrine or clonidine were counteracted by yohimbine, a preferential blocker of alpha-2 receptors, but not by prazosine, an alpha-1 receptor blocker. The glucose-stimulated uptake of 45Ca2+ in the islets was only consistently inhibited by epinephrine. This effect was counteracted by yohimbine. Clonidine had no effect on the release of 86Rb+ from preloaded islets. It is concluded that insulin secretion is suppressed by alpha-2 receptor agonism in the pancreatic beta-cells and that this effect is mediated by mechanisms other than the transmembrane fluxes of calcium or potassium ions.

Dissociation of beta-adrenoceptor-induced effects on amylase secretion and cyclic adenosine 3', 5' monophosphate accumulation.

By using a multi-channel microperifusion system the effects of noradrenaline, the beta1-adrenoceptor agonist prenalterol, and the beta2-selective agonist terbutaline were studied on amylase pig submandibular glands. 2 Noradrenaline caused significant amylase discharge and cyclic AMP accumulation. 3 Prenalterol was as effective as noradrenaline in causing amylase release but did not significantly affect the cyclic AMP content. 4 Terbutaline stimulated cyclic AMP accumulation, but had little effect on amylase secretion. 5 The present study reveals that there is a dissociation of the beta-adrenoceptor-induced amylase release and cyclic AMP formation, and that this dissociation may be due to different beta-adrenoceptor subtypes.

Effects of neonatal sympathetic denervation on amylase secretion in the adult rat parotid gland: difference in beta 1- and beta 2-adrenoceptor response.

Changes in amylase secretion and cyclic AMP accumulation in response to various secretagogues were studied in parotid glands of adult rats subjected to neonatal sympathetic denervation by unilateral excision of the superior cervical ganglion. Denervation decreased the gland content of amylase and both basal and the stimulated levels of cyclic AMP were elevated. The secretory cells of neonatally denervated glands exhibited enhanced maximal enzyme discharge in response to beta-adrenoceptor agonists. However, the selective beta 1-agonist, prenalterol was not effective in this respect whereas an enhanced maximal secretory response to the beta 2-selective agonist, terbutaline, was particularly prominent. DBcAMP was also more efficient in inducing amylase release from the denervated gland. The result of the present study demonstrate that the usual dominance of the beta 1-adrenoceptor subtype in eliciting amylase release is lost, implying that the differentiation of the beta-adrenoceptor into its subtypes is altered by neonatal sympathetic denervation.

Characterization of the rat parotid beta-adrenoceptor.

1 The effects of various beta-adrenoceptor agonists on amylase secretion from the rat parotid gland were studied by means of two different in vitro techniques. 2 The dose-response relation for each agonist was established, as also were the ED50 values. 3 All drugs appeared to act directly on the acinar cells, as reserpine-treatment did not abolish their secretagogic effects. 4 Two groups of agonists could be distinguished: one group consisting of adrenaline, noradrenaline and the B1-selective agonist prenalterol (H133/22) with a high enzyme discharge potency and a second group consisting of the beta 2-agonists, terbutaline and salbutamol, with a markedly lower effect. 5 The present data further support the theory that rat parotid acinar cells are supplied mainly with beta-adrenoceptors of the beta 1-subtype, similar to those present in heart and adipose tissue.

Cytotoxic activation of complement by mouse pancreatic islet cells.

Human serum, or serum proteins excluded by Sephadex G-25, irreversibly inhibited the ability of mouse pancreatic islet cells to accumulate Rb+. The same treatment reduced the capacity of serum to subsequently inhibit Rb+ uptake by fresh islet cells or to lyse sensibilized sheep erythrocytes. Serum-treated islet cells exhibited electron microscopic signs of damage, including ruptures of the plasma membrane, swelling of mitochondria, and reduced electron density of the cytoplasmic ground substance. Serum induced a prompt insulin release, which was not inhibited by epinephrine. The serum effects were prevented by mild heating (50 degrees C or 56 degrees C, 30 min) but not by treating serum with 10 mM EGTA and 10 mM MgCl2, or with soybean trypsin inhibitor. Inhibition of Rb+ accumulation in response to human serum was also observed with dispersed mouse exocrine pancrease, liver, and spleen cells but not with whole islets. Homologous mouse serum had no effect on mouse liver or spleen cells but significantly decreased the Rb+ uptake by mouse islet cells. Autologous serum had no noticeable effect. It is suggested that mouse islet cells can activate complement via the alternative pathway and that triggering of this pathway is controlled by cellular discriminators of species, organ, and self.

Initial uptake and insulin releasing action of chloromercuribenzene-p-sulphonic acid (CMBS) in suspensions of pancreatic islet cells.

The effects of chloromercuribenzene-p-sulphonic acid on dispersed cells prepared from beta-cell-rich ob/ob-mouse islets were studied. 1) Chloromercuribenzene-p-sulphonic acid at concentrations of 0.1 mmol/l or higher diminished cell viability which was partially counteracted by increasing concentrations of bovine serum albumin. 2) The uptake of 203Hg-chloromercuribenzene-p-sulphonic acid after incubation for 4 seconds or longer showed that most of the non-toxic concentrations of chloromercuribenzene-p-sulphonic acid was bound to the cell within 40 seconds. Maximal uptake was achieved after 3 minutes of incubation. The uptake of radioactive chloromercuribenzene-p-sulphonic acid was inhibited by bovine serum albumin. 3) The dynamics of insulin release from perifused dispersed beta-cells embedded in fibrin showed a maximal 40--50-fold stimulation by 0.03 mmol/l chloromercuribenzene-p-sulphonic acid within 10 minutes of perifusion. 4) Scanning electron microscopy of beta-cells revealed no major changes in the cell surface under conditions of maximal binding and insulin releasing effects of chloromercuribenzene-p-sulphonic acid. These data support the concept that the ability of chloromercuribenzene-p-sulphonic acid to induce insulin release is related to its initial binding to the beta-cell surface. The binding of chloromercuribenzene-p-sulphonic acid and the subsequent release of insulin seem to occur without major changes in beta-cell surface morphology.