Evidence for an inhibitor of insulin release in the pancreatic islets

Summary The release of insulinin vitro from isolated mouse islets was significantly inhibited in the presence of an islet protein extract equivalent to more than 100 times the normal serum level of insulin. The nature of the inhibitory islet substance remains unclear. The possibility that the blood circulation through the islets may be important for the local regulation of insulin release by reducing high levels of this hormone in the immediate surroundings of the cells should be considered.Part of a project supported by the United States Public Health Service (AM-12535), Swedish Medical Research Council (12×-562) and the Medical Faculty of Umeå.     

Effect of human lymphoblastoid interferon on insulin synthesis and secretion in isolated human pancreatic islets

Summary Human islets of Langerhans were isolated from the pancreas removed from a 13-year-old female transplant donor. The islets were incubated in a culture medium for 24 h in the presence of human lymphoblastoid interferon (1000 units/ml). Insulin secretion, proinsulin biosynthesis, total protein biosynthesis and total insulin content were assessed at various concentrations of glucose in the presence of interferon. In interferon-treated islets glucose-stimulated insulin secretion was unaltered from that of control islets; however, glucose-stimulated proinsulin biosynthesis was specifically inhibited by interferon (48%, p<0.025). Total protein biosynthesis and total insulin content were not significantly affected by interferon.     

Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets

Aims/hypothesis. The role of beta-cell metabolism for generation of oscillatory insulin release was investigated by simultaneous measurements of oxygen tension (pO₂) and insulin release from individual islets of Langerhans.¶Methods. Individual islets isolated from the ob/ob-mice were perifused. Insulin in the perifusate was measured with a sensitive ELISA and pO₂ with a modified Clark-type electrode inserted into the islets.¶Results. In the presence of 3 mmol/l d-glucose, pO₂ was 102 ± 9 mmHg and oscillatory (0.26 ± 0.04 oscillations/min). Corresponding insulin measurements showed oscillatory release with similar periodicity (0.25 ± 0.02 oscillations/min). When the d-glucose concentration was increased to 11 mmol/l, pO₂ decreased by 30 % to 72 ± 10 mmHg with maintained frequency of the oscillations. Corresponding insulin secretory rate rose from 5 ± 2 to 131 ± 16 pmol · g^–1· s^–1 leaving the frequency of the insulin pulses unaffected. The magnitude of glucose-induced change in pO₂ varied between islets but was positively correlated to the amount of insulin released (r ² = 0.85). When 1 mmol/l tolbutamide was added to the perifusion medium containing 11 mmol/l glucose no change in average oscillatory pO₂ was observed despite a doubling in the secretory rate. When 8 mmol/l 3-oxymethyl glucose was added to perifusion medium containing 3 mmol/l d-glucose, neither pO₂ nor insulin release of the islets were changed. Temporal analysis of oscillations in pO₂ and insulin release revealed that maximum respiration correlated to maximum or close to maximum insulin release.¶Conclusion/interpretation. The temporal relation between oscillations in pO₂ and insulin release supports a role for metabolic oscillations in the generation of pulsatile insulin release. [Diabetologia (2000) 43: 1313–1318]     

Evidence for a circadian rhythm of insulin release from perifused rat pancreatic islets

Summary This study aims to analyse a circadian rhythm of insulin secretion from isolated rat pancreatic islets in vitro and its potential modulation by melatonin, the concentrations of which change in vivo inversely to that of insulin. The circadian rhythm was evaluated in a perifusion system, adapted to the specific conditions of pancreatic islets. To determine rhythmicity of insulin secretion, 30-min fractions were collected continuously for investigative periods of 44 to 112 h. Insulin secretion in 10 experiments was analysed by using the MacAnova-program for period length (τ), the χ²-periodogram for test of significance (p < 0.001), and additionally the empirical cosine adaptation for amplitude and goodness-of-fit. Thereby a circadian pattern was observed with periods (τ) between 21.8 and 26.2 h. The period duration (mean ± SEM) was 23.59 ± 0.503 h, the overall mean insulin release 1038 ± 13 pmol/l and the mean amplitude 88 ± 17 pmol/l. Adding melatonin (10 nmol/l, t = 2 h) as a hormonal Zeitgeber during analysis of circadian insulin secretion phase-response studies show phase-shifts with approximately 9 h phase advance. Thereafter the circadian period was maintained, while the amplitude was enhanced. From this it is concluded that an endogenous circadian oscillator is located within the pancreatic islets of the rat that regulates circadian insulin secretion of the insulin-producing beta cells. The pacemaker is remarkably stable, because its periodicity is not affected by factors altering insulin secretion. In agreement with inhibitory influences of melatonin (range 0.5 nmol/l to 5 μmol/l) on the insulin response in vitro, the phase-responses support the contention that pancreatic beta cells may be targets for melatonin action. [Diabetologia (1998) 42: 1085–1092] Received: 11 December 1997 and in final revised form: 20 March 1998     

Daily rhythm of glucose-induced insulin secretion by isolated islets from intact and pinealectomized rat

It is well known that pinealectomy induces in rats a diminished glucose tolerance, insulin resistance, a reduction in GLUT4 content in adipose and muscular tissues, a decrease in hepatic and muscular glycogenesis, impairment of glucagon action and an increase in blood pyruvate concentration. In addition, it has been shown that melatonin suppresses insulin secretion in several experimental conditions. The objective of the present study was to investigate the daily rhythm of glucose-induced insulin secretion and glucose oxidation by isolated pancreatic islets and to investigate the effect of chronic absence of melatonin (30 days of pinealectomy) on this rhythmic process. The data obtained confirmed the presence of a strong 24-hr rhythm of insulin secretion by isolated pancreatic islets. In addition, it was demonstrated that the glucose-metabolizing ability of the B-cell follows a daily rhythm phase locked to insulin secretion rhythm. Most interesting, however, was the demonstration that the daily rhythmic processes of insulin secretion and B-cell -[U-14C]-glucose oxidation by isolated pancreatic islets is completely modified by the chronic absence of the pineal gland. Thus, pinealectomy induced in all groups an increase in 24-hr mean glucose-stimulated insulin secretion and [U-14C]-glucose oxidation, in addition to some alterations in the rhythmic amplitude and a remarkable phase-advancing of the daily curves for 8.3 mm glucose (a condition similar to that observed in fed animals and where the B-cells are supposedly more active). These observations strongly suggest that the presence of the pineal gland may be necessary for the proper synchronization of these metabolic rhythms with other circadian rhythms like activity-rest and feeding.     

Role of infiltrating T cells for impaired glucose metabolism in pancreatic islets isolated from non-obese diabetic mice

Summary Pancreatic islets isolated from non-obese diabetic (NOD) mice, all of which have insulitis, exhibit an impaired glucose metabolism. In order to investigate the role of infiltrating lymphocytes for this altered metabolism, we injected 12- to 13-week-old female NOD mice with monoclonal antibodies directed against either the -T cell receptor, CD4⁺ or CD8⁺ T cells. Control NOD mice were injected with normal rat IgG or with the vehicle (phosphate buffered saline) alone. Injection of the three different monoclonal antibodies markedly reduced the mononuclear cell infiltration. An intravenous glucose tolerance test showed no differences between the groups. Islet insulin release in response to glucose was similar in all groups. In contrast, islets isolated from the control NOD mice with insulitis showed a high basal (1.7 mmol/l glucose) glucose oxidation rate and a small increase in the glucose oxidation rate in response to a high glucose concentration (16.7 mmol/l glucose). The monoclonal antibodies counteracted the elevated basal glucose oxidation rate of the islets. Parallel studies of stimulated mononuclear cells suggested that the contribution of glucose oxidized by islet-infiltrating lymphocytes could only partially explain the observed alterations in NOD mouse islet metabolism. Culture of islets obtained from NOD mice in the presence of the cytokine interleukin-1 induced a similar pattern of glucose metabolism as seen earlier in IgG or phosphate-buffered saline treated control NOD mice. In conclusion, alterations in the glucose oxidation rates seem to be an early sign of disturbance in islets isolated from NOD mice. These early alterations in glucose metabolism can be reversed in vivo by monoclonal antibodies directed against effector lymphocytes. This suggests that the infiltrating mononuclear cells can induce reversible alterations in pancreatic Beta-cell function which may precede impaired insulin secretion, Beta-cell destruction and overt diabetes mellitus.     

Normoglycaemia after transplantation of freshly isolated and cryopreserved pancreatic islets in Type 1 (insulin-dependent) diabetes mellitus

Summary Purified islets of Langerhans and a kidney were transplanted into a 36-year-old patient who suffeded from renal failure secondary to a 25 year history of Type 1 (insulin-dependent) diabetes mellitus. The islet graft contained 243 000 fresh islets (mean islet diameter 150 m) that were syngeneic with the kidney fraft and 368 000 cryopreserved islets that had been collected from four other donors. The total of 10 000 islets/kg body weight was infused into the liver via the umbilical vein. Immunosupperession was induced with antilymphocyte globulin and maintained with prednisone, cyclosporine and azathioprine. Serum C-peptide levels (ng/ml) during fasting and after standard mixed metal feeding (Sustacal) were <0.12 preoperatively. Postoperatively, insulin secretion was restored: fasting C-peptide rose during the first 4 weeks to levels of 4 to 5 and Sustacal elicited a further rise to 6 to 7. Transplant renal function was stable. Dialy fasting glucose (mmol/l, mean±SD) was 5.6±1 and 5.3±0.6 during the first and second months respectively and post-Sustacal glucose was 5.7+-0.8. Exogenous insulin therapy was progressively withdrawn and stopped duting the ninth week. Thereafter, fasting glucose was 4.7+-0.5, 24 h mean glucose was 6.6+-0.5, and normoglycaemia was maintained after Sustacal. These data show that this mass of freshly isolated and cryopreserved islets from multiple donors provided sustained function (3 months) that reversed insulin-dependence in an immunosuppressed Type 1 diabetic patient treated with simultaneous islet-kedney transplantation.     

Tolbutamide stimulation and inhibition of insulin release: Studies of the underlying ionic mechanisms in isolated rat islets

Summary The effects of tolbutamide on insulin release, ⁴⁵Ca²⁺ uptake and ⁸⁶Rb⁺ efflux were studied in isolated rat islets. At a low glucose concentration (75 mg/dl), tolbutamide (20–500 g/ml) produced a rapid, dose-dependent increase in insulin release from perifused islets. After 30–40 min however, the rate of secretion as well as the potentiating effect of theophylline were inversely related to the concentration of sulphonylurea. The monophasic release of insulin triggered by tolbutamide (100 g/ml) at low glucose could be evoked again by removing and reintroducing the drug, or by temporarily withdrawing calcium or adding cobalt to the medium. Tolbutamide (20 g/ml) accelerated and potentiated the biphasic insulin release in response to a secondary stimulation by glucose (150 mg/dl). By contrast, 100 g/ml tolbutamide reduced the releasing effect of glucose to a slow increase in secretion rates. Theophylline normalized the second phase of release, but did not restore the rapid phase. Tolbutamide stimulated ⁴⁵Ca²⁺ influx (2 min-uptake) in islet cells; this effect was maximum immediately after addition of the drug and decreased later on, exhibiting a monophasic pattern. Glucose stimulation of Ca²⁺ uptake (5 min) was reduced in the presence of 100 g/ml tolbutamide. At a low glucose concentration, tolbutamide reversibly reduced ⁸⁶Rb⁺ efflux (tracer of K⁺) from islet cells, without altering the further inhibition of this efflux by a later glucose increase. It is suggested that tolbutamide depolarizes B cells partially by reducing their K⁺ permeability. This depolarization leads to opening of voltagedependent calcium channels and the resulting Ca²⁺ influx triggers insulin release. The important and maintained depolarization by high concentrations of tolbutamide may secondarily inactivate these channels and cause a decrease in Ca²⁺ influx. This could explain the monophasic release of insulin and the refractoriness of B cells to subsequent glucose stimulation.     

The effect of biguanides on secretion and biosynthesis of insulin in isolated pancreatic islets of rats

Summary Based on the clinical observation that biguanide treatment of obese patients may alter insulin levels, the influence of metformin and phenformin on basal and glucose stimulated insulin secretion, as well as on insulin biosynthesis, was studied in isolated islets of rats. — Biguanide concentrations of 100 g/ml, or higher, significantly reduced glucose stimulated insulin secretion. Both dose dependence and a difference in the intrinsic activities of metformin and phenformin were demonstrated. Incubating the same islets for a second period without biguanides, glucose stimulated insulin secretion was still decreased. Addition of glibenclamide during this second period increased insulin secretion, but did not overcome complete inhibition achieved after incubation at very high biguanide concentrations. Glucose stimulated biosynthesis of proinsulin and insulin was decreased in the presence of biguanides and completely suppressed at very high concentrations. Inhibition of cell respiration in the islet cells effected by high biguanide doses may be the reason for the inhibition of secretion and biosynthesis of insulin. — On the other hand, an insulin release was found at the highest phenformin concentration of 10 mg/ ml and during perfusion of the isolated rat pancreas with higher biguanide doses. — Biguanide concentrations found to be effective in this study are very high compared with therapeutic levels. Moreover, biguanide actions are known to be highly dependent on species, concentration and metabolic situation. — Definite conclusions from these findings regarding clinical significance, therefore, seem unwarranted.Supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Oxidation of glucose and fatty acids in normal and in A2-cell rich pancreatic islets isolated from guinea-pigs

Summary Glucose and fatty acid oxidation has been measured in normal guinea-pig islets of Langerhans, and in A₂-cell rich islets from streptozotocin-treated guinea-pigs. The rate of oxidation of these compounds in guinea-pig A₂-cells and B-cells has been estimated. In the B-cells, the oxidation of glucose and octanoic acid responded markedly to changes in the extracellular levels of these substrates. Palmitic acid did not appear to be oxidized by the B-cells. In contrast, the oxidation of octanoic acid and palmitic acid in the A₂-cells was very sensitive to changes in the extracellular fatty acid concentration. The sensitivity of glucose oxidation to changes in the glucose concentration was small by comparison. The high rate of oxidation of fatty acids in the A₂-cells supports the view that the rate of fatty acid metabolism in these cells plays an important role in the regulation of glucagon release.

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Die Oxidation von Glucose und Fettsäuren in normalen und isolierten, mit A₂-Zellen angereicherten Pankreasinseln vom Meerschweinchen

Zusammenfassung An normalen Langerhansschen Inseln vom Meerschweinchen und an mit A₂-Zellen angereicherten Inseln von streptozotozin-behandelten Meerschweinchen wurde die Oxidation von Glucose und Fettsäuren gemessen und die Oxidationsrate in A₂-Zellen und B-Zellen des Meerschweinchens bestimmt. In den B-Zellen hing die Oxidation von Glucose und Octansäure stark von den Änderungen der extracellulären Konzentrationen dieser Substanzen ab. Palmitinsäure schien in den B-Zellen nicht oxidiert zu werden. Dagegen war die Oxidation von Oktansäure und Palmitinsäure in den A₂-Zellen sehr von den Schwankungen der extracellulären Fettsäurekonzentration abhängig. Die Änderung der Glucoseoxidation bei Schwankungen der Glucosekonzentration war im Verhältnis dazu gering. Die hohe Oxidationsrate der Fettsäuren in den A₂-Zellen unterstützt die Theorie, daß der Fettsäuremetabolismus dieser Zellen eine wesentliche Rolle in der Regulierung der Glucose-sekretion spielt.

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Oxydation du glucose et des acides gras dans les îlots pancréatiques normaux et dans les îlots riches en cellules A₂ prélevés sur le cobaye

Résumé L'oxydation du glucose et des acides gras a été mesurée dans les îlots normaux de Langerhans chez le cobaye, ainsi que dans les îlots riches en cellules A₂ du cobaye traité à la streptozotocine. Le taux d'oxydation de ces composés dans les cellules A₂ et les cellules B du cobaye a été estimé. Dans les cellules B, l'oxydation du glucose et de l'acide octanoique a fortement répondu à des changements dans les taux extra-cellulaires de ces substrats. L'acide palmitique ne semble pas avoir été oxydé par les cellules B. Au contraire, l'oxydation de l'acide octanoique et de l'acide palmitique dans les cellules A₂ était très sensible aux changements dans la concentration des acides gras extra-cellulaires. La sensibilité de l'oxydation du glucose envers les changements dans la concentration du glucose était comparativement faible. Le taux élevé de l'oxydation des acides gras dans les cellules A₂ corrobore l'hypothèse que le degré du métabolisme des acides gras dans ces cellules joue un rôle important dans la régulation de la sécrétion du glucagon.

     

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (∼2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]_i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]_i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]_i and then potentiate glucose-induced insulin secretion and production in parallel.     

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (～2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]_i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]_i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]_i and then potentiate glucose-induced insulin secretion and production in parallel.     

Endothelin-1 stimulates insulin secretion by direct action on the islets of Langerhans in mice

Summary Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, is secreted in response to insulin. Elevated circulating ET-1 levels have been found in patients with diabetes mellitus and vascular dysfunction. The question arises whether ET-1 acts as a direct modulator of insulin secretion. To test this, we studied the effects of ET-1 on isolated mouse islets of Langerhans. ET-1 (1 nmol/l-1 mol/l) dose-dependently stimulated insulin secretion from islets incubated in the presence of 16.7 mmol/l glucose (p<0.05). The effect of ET-1 is glucose-dependent since no potentiation was found at 3.3 mmol/l glucose. Furthermore, ET-1 induced a large, transient increase in glucose-stimulated insulin secretion during islet perifusion in the presence (p<0.001), but not in the absence, of extracellular Ca²⁺. The rate of ⁴⁵Ca²⁺-efflux from ⁴⁵Ca²⁺-prelabelled islets was transiently stimulated by ET-1 during perifusion at 16.7 mmol/l glucose in the presence of extracellular Ca²⁺ (p<0.001). A short-lived increase in ⁴⁵Ca²⁺-efflux was also observed in the absence of extracellular Ca²⁺ (p<0.05). It is suggested that the effects of ET-1 on insulin secretion are critically dependent on influx via Ca²⁺-channels. In addition, ET-1 transiently enhanced ⁸⁶Rb⁺-efflux from ⁸⁶Rb⁺-prelabelled islets both in the presence (p<0.001) and in the absence (p<0.001) of extracellular Ca²⁺ suggesting that ET-1 does not elicit insulin secretion by inhibition of the potassium permeability. Our study provides evidence that ET-1 stimulates insulin secretion via a direct effect on the islets of Langerhans.Abbreviations ET-1 Endothelin-1     

Properties of isolated human islets of langerhans: insulin secretion,glucose oxidation and protein phosphorylation

Summary In the present study, human islets were isolated by collagenase digestion from the pancreases of three kidney donors. Maintainance of the islets in tissue culture enabled insulin release, glucose oxidation and Ca²⁺-calmodulin-dependent protein phosphorylation to be determined using the same islets. Increasing glucose over a range 0–20 mmol/l resulted in a sigmoidal stimulation of insulin release (28.8±5.2 to 118.4±25.8 U-islet^–-h^–, n=10; threshold <4 mmol/l). There was a marked correlation between the insulin secretory response of the islets to glucose and their rate of glucose oxidation (5.9±0.3 at glucose 2 mmol/l up to 25.8±1.8 pmol-islet^–.h^– at 20 mmol/l, r = 0.98). N-acetylglucosamine (20 mmol/l) failed to elicit a secretory response from the islets. Stimulation of insulin secretion by glucose was dependent upon the presence of extracellular Ca²⁺. Extracts of the islets contained a Ca²⁺-calmodulin-dependent protein kinase which phosphorylated a 48-kdalton endogenous polypeptide. Myosin light-chain kinase activity was demonstrated in the presence of exogenous myosin light chains. This report demonstrates for the first time the sigmoidal nature of glucose-stimulated insulin release from isolated human islets, and its correlation with enhanced glucose oxidation. Furthermore, this is the first report of the presence of Ca²⁺-dependent protein kinases in human islets.     

Dynamics of O2 consumption in rat pancreatic islets

Summary The O₂ consumption of rat pancreatic islets was determined by monitoring pO₂ in the perifusate from groups of 200–300 islets. Basal respiration was maintained for up to 2 h. The insulin secretagogues, glucose and 4-methyl-2-oxopentanoate, provoked an immediate (<5 s) increase in islet respiration which attained a new steady-state within 10–40 min. The respiratory changes were immediately reversible upon removal of the substrate and were parallelled by changes in insulin release and substrate oxidation. The concentration dependence of glucose-induced respiratory changes was sigmoidal with a threshold at 3 mmol/l. The concentration dependence with 4-methyl-2-oxopentanoate was characterised by a hyperbolic relationship. The weak insulin secretagogues 3-methyl-2-oxobutyrate and d,1-3-methyl-2-oxopentanoate, although stimulating islet respiration were not more effective than 4-methyl-2-oxopentanoate at non-insulinotropic concentrations. Rotenone, antimycin and oligomycin inhibited both basal O₂ consumption and the ability of glucose and 4-methyl-2-oxopentanoate to increase islet respiration. 2,4-Dinitrophenol increased islet O₂ consumption. The omission of Ca²⁺ and Mg²⁺ from the perifusing media, or the addition of the ionophore A23187, had little effect on respiration. The omission of K⁺ inhibited glucose-induced changes but had a lesser effect in the absence of substrate or in the presence of 4-methyl-2-oxopentanoate. The omission of HCO₃ ^- reduced both basal and secretagogue-induced changes in islet respiration. It is concluded that mitochondrial O₂ consumption linked to oxidative phosphorylation is a major component in the respiratory response, and that some energy consuming process in the islets depends on the availability of HCO₃ ^-. Mitochondrial reactions may generate a signal initiating the secretory process.     

Evidence for priming and inhibitory effects of glucose on insulin secretion from isolated islets of langerhans

Summary Biphasic insulin secretion from perifused rat islets of Langerhans was affected in three ways by the islet glucose environment prior to stimulation, (i) The secretory response to glucose was diminished if the basal concentration of glucose in the medium was reduced from 5.5 to 2.7 mmol/l for 2 h prior to stimulation. First phase secretion was affected more than the second, (ii) Secretion was potentiated if islets had been previously exposed to a stimulatory concentration of glucose of 22.2 mmol/l. Again first phase secretion was particularly affected and there was a positive correlation between the magnitude of the secretory response and the duration of the initial stimulus, (iii) In contrast, both phases of secretion were proportionately reduced if islets had been previously exposed to stimulatory concentrations of glucose of 8.3 mmol/l.     

Myocardial electrophysiological properties in the presence of an AT1 angiotensin II receptor antagonist

Introduction Blockade of the AT₁ angiotensin II (Ang II) receptor has been shown to provide antihypertensive effects. However, whether AT₁ Ang II receptor antagonists influence myocardial electrophysiological properties remains unclear.Methods and results Accordingly, atrial and ventricular myocardial electrophysiological properties were examined in adult rat (n=13) and guinea pig (n=9) myocardial preparations in the presence of the specific AT₁ Ang II receptor antagonist, valsartan (CGP 48933; 0.5, 5, or 500 mol/L). These concentrations reflect up to 100 fold higher drug concentrations than those observed in clinical trials. Transmembrane potential data were recorded using standard microelectrode techniques at baseline and following superfusion with valsartan. The lower concentrations of valsartan (0.5 and 5 mol/L) had minimal effects on myocardial electrophysiology. In the presence of 500 mol/L of valsartan, resting membrane potential increased from baseline in both rat (–82.3±4.1 vs –76.8±5.8 mV, p<0.05) and guinea pig (–81.6±2.9 vs –76.9±2.0 mV, p<0.05) atrial myocardium. Action potential duration at 90% repolarization was increased in guinea pig atrial (91.7±1.4 vs 80.0±5.6 ms, p<0.05) and ventricular (131.1±8.1 vs 118.7±8.3 ms, p<0.05) myocardium following exposure to 500 mol/L of valsartan. In a separate series of experiments Ang II (1.0 mol/L) had no effect on atrial or ventricular action potential characteristics in either species.Conclusion Thus, the effects of valsartan, which were observed only at concentrations 100 fold higher than those reported in clinical trials, may be due to non-specific drug interactions with the myocyte sarcolemma.     

Evidence that somatostatin inhibits proinsulin synthesis and insulin release by isolated pancreatic islets of the rat

Conflicting reports on the direction and magnitude of the effect of somatostatin on (pro)insulin synthesis prompted our investigation. Two assays for proinsulin synthesis were designed in which [4,5-3H]-L-leucine incorporation into proinsulin was normalized on the basis of postincubation insulin levels rather than on the number of islets incubated. Somatostatin at a concentration of 10 micrograms/ml inhibited 300 mg/dl glucose-stimulated proinsulin synthesis by 25% from 448 +/- 25 dpm/microunits insulin to 336 +/- 25 dpm/microunits insulin (disintegrations per minute in the proinsulin peak per microunit extractable insulin) (p less than 0.05). Glucagon (10 micrograms/ml) reversed the inhibitory effect of somatostatin on proinsulin synthesis from 336 +/- 25 dpm/microunits insulin to 480 +/- 44 dpm/microunits insulin (p less than 0.02). Somatostatin (10 micrograms/ml) had no significant effect on proinsulin synthesis in the presence of 70 mg/dl or 150 mg/dl glucose. Insulin release in 300 mg/dl glucose was inhibited 38% by 10 micrograms/ml somatostatin from 3.05 +/- 0.40 mU medium/mU tissue to 1.90 +/- 0.10 mU medium/mU tissue (p less than 0.01) over a 45-minute incubation period. These data suggest that somatostatin may act on glucose signal transduction on a level at which both insulin synthesis and secretion are affected. Further, the results are consistent with the hypothesis that cyclic AMP participates in mediating somatostatin effects on B-cell metabolism.