Stimulatory effect of serum from diabetic patients on insulin release from mouse pancreatic islets maintained in tissue culture

Summary Islets of Langerhans from NMRI-mice were kept for one week in tissue culture in medium supplemented with human serum obtained from either normal healthy subjects or newly diagnosed juvenile diabetic patients before insulin treatment. Islets cultured in diabetic serum released more inslin than islets cultured in normal serum, whether tissue culture medium 199 with 5.5–8.3 mmol/1 glucose and 10% serum, or culture medium RPMI1 640 with 11 mmol/1 glucose and 0.5% serum were used. Islets kept for one week in culture with diabetic serum did not show any decrease in DNA content or glucose induced insulin secretion and biosynthesis. It is concluded that serum from newly diagnosed insulin dependent diabetic patients stimulates insulin release from isolated mouse islets kept in tissue culture. The underlying mechanism is unknown.     

Effects of 3-isobutyl-1-methylxanthine on neonatal pancreatic islets maintained in tissue culture

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) promoted the formation of monolayers in cultured pancreatic islets isolated from neonatal rats. Immunofluorescence with specific antisera to insulin and glucagon revealed B-cells and A-cells in these monolayers. Glucose-mediated insulin release was increased by raising the glucose concentration from 5 to 10 mmoles/l. Addition of IBMX (0.1 mmoles/l) to medium containing 10 moles/l glucose produced a further increase in insulin release. Recovery of total insulin, i.e. intracellular insulin plus insulin secreted, was also increased by approximately 50% after 8 days of culture. The B-cells showed a marked biosynthetic response to an acute glucose challenge after prior culture with 10 mmoles/l glucose. Although both unstimulated (1.5 mmoles/l glucose) and stimulated rates (1.5 mmoles/l glucose) of [3H]leucine incorporation into (pro)insulin were significantly higher following culture in 10 mmoles/l glucose plus IBMX (0.1 mmoles/l) than after prior culture with 10 mmoles/l glucose alone, the percentage of (pro)insulin synthesized in relation to total protein synthesis was only increased at the low concentration of glucose. These studies demonstrate that monolayer cultures of neonatal B-cells can be readily produced by IBMX and maintained in a functional state, as defined by their secretory and biosynthetic response. It is suggested that the phosphodiesterase inhibitor exerts a sensitizing effect on the responsiveness of the B-cell to glucose. Moreover, the culture system employed in the present study may prove to be useful for further studies of various agents affecting the B-cell function.     

Effects of L-leucine on the insulin production,oxidative metabolism and mitochondrial ultrastructure of isolated mouse pancreatic islets in tissue culture

Summary This study was performed to evaluate whether L-leucine is able to relieve the structural and functional alterations previously described in pancreatic islets exposed in vitro for a prolonged time to a subnormal glucose concentration (3.3 mM). It was found that both the impairment of secretion and the decreased rate of biosynthesis of insulin characteristic, of islets cultured for one week in 3.3 mM glucose were prevented by adding 15 mM L-leucine to the culture medium. Furthermore, the rates of tritiated water production and glucose or leucine oxidation were significantly enhanced after culture in the presence of L-leucine. The rate of DNA synthesis as estimated by the incorporation of tritiated thymidine was, however, unchanged by the presence of L-leucine in the culture medium. Leucine cultured islet cells displayed ultrastructural signs of high functional activity. A detailed morphometric examination revealed fewer but hypertrophic mitochondria. The present results suggest that L-leucine can replace glucose in several respects as a long-term stimulus of the pancreatic B-cells, possibly by acting as a metabolic substrate.Presented in part at the Congresses of the European Association for the Study of Diabetes, Munich, 1975 and Helsinki, 1976     

Culture of mouse pancreatic islets in different glucose concentrations modifies B cell sensitivity to streptozotocin

Summary There have previously been divergent data published regarding the effects of glucose on the diabetogenic effects of streptozotocin. In order to further explore this issue, two separate sets of experiments were performed. In the first, mouse pancreatic islets were maintained in culture for 3 days at different glucose concentrations (5.6,11.1 and 28 mmol/l) and then exposed to streptozotocin. After another 3 days in culture at 11.1 mmol/l glucose, the B cell function was evaluated by measurement of glucose-stimulated insulin release, the number of islets recovered after culture, and the islet DNA and insulin contents. In the second group of experiments islets were first maintained in culture at 11.1 mmol/l glucose, then treated with streptozotocin and subsequently cultured for 6 days at the different glucose concentrations given above. It was found that islets maintained in a medium containing 28 mmol/l glucose before or after streptozotocin exposure showed less signs of damage than islets cultured in 11.1 mmol/l glucose. A similar, but less pronounced, de creased sensitivity to streptozotocin was found in islets precultured in 5.6 mmol/l glucose, in comparison with those islets cultured in 11.1 mmol/l glucose. Culture at 5.6 mmol/l glucose just after streptozotocin treatment did not induce any improvement in islet survival or function. It is suggested that the increased damage induced by streptozotocin to islets precultured at 11.1 mmol/l glucose, in comparison with 5.6 mmol/l glucose, can be related to the fact that an increased metabolic activity of B cells render them more susceptible to the toxin. The improved preservation of islets cultured at 28 mmol/l glucose before or after streptozotocin treatment may reflect an additional effect of glucose, i. e. activation of defense mechanisms in the B cells against cytotoxins.     

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.     

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.     

Hypophysis and function of pancreatic islets

Summary Insulin secretion and biosynthesis of proinsulin and insulin were determined in isolated pancreatic islets of hypophysectomized rats. Control rats were of both same age and weight. Hypophysectomy was performed either 13 or 5 weeks prior to the investigation, the weight of the animals being either 80 or 170 g. Biosynthesis of insulin was estimated from the amounts of radioactivity incorporated into proinsulin and insulin after incubation of isolated islets at 50 or 300 mg% glucose in the presence of³H-leucine for 3 h. Islet proteins were separated on Sephadex G 50 fine. — Hypophysectomy resulted in a significant decrease of both glucose stimulated secretion and biosynthesis of insulin. It was found that this reduction was 1) more significant when compared with controls of same age 2) more marked in rats which had been hypophysectomized 13 weeks before than in rats after an interval of 5 weeks and 3) less in rats which had been hypophysectomized at a weight of 170 g than in rats in whom pituitary ablation was performed at a weight of 80 g. At basal glucose concentrations, no significant changes of both secretion and biosynthesis of insulin were apparent. The relation of radioactivity incorporated into proinsulin and insulin was unchanged under all conditions. Insulin content of the isolated islets used was found within about the same range in all rats, apart from the animals which had been hypophysectomized 13 weeks before. In islets of these rats, a reduction to 84% was observed. — Our findings may be explained by reduced sensitivity of the pancreatic B-cell to glucose and a slower rate of insulin biosynthesis after hypophysectomy.Alexander von Humboldt-Fellow 1970–1972.H.S. was on leave from the 2nd Medical Clinic, University of Vienna, Austria.Supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, SFB Ulm 87, Proj. 11.     

Inhibitory effect of kisspeptins on insulin secretion from isolated mouse islets

Islet hormone secretion is regulated by a variety of factors, and many of these signal through G protein-coupled receptors (GPCRs). A novel islet GPCR is GPR54, which couples to the Gq isoform of G proteins, which in turn signal through the phospholipase C pathway. Ligands for GPR54 are kisspeptins, which are peptides encoded in the KISS1 gene and also expressed in islet β-cells. The KISS1 gene encodes a hydrophobic 145-amino acid protein that is cleaved into a 54-amino acid protein, kisspeptin-54 or KP54. Shorter kisspeptins also exist, such as kisspeptin-10 (KP10) and kisspeptin-13 (KP13). The involvement of GPR54 and kisspeptins in the regulation of islet function is not known. To address this problem, we incubated isolated mouse islets in the presence of KP13 and KP54 for 60 min and measured insulin secretion. We found that both KP13 and KP54 at 10 nM, 100 nM and 1μM inhibited insulin secretion in the presence of 2.8 mM glucose. However, by increasing the glucose concentration, this inhibitory action of the kisspeptins vanished. Thus, at 11.1 mM glucose, KP13 and KP54 inhibited insulin secretion only at high doses, and at 16.7 mM they no longer inhibited insulin secretion in any of the doses. We conclude that kisspeptins inhibit insulin secretion at glucose concentrations below 11.1 mM. This suggests that kisspeptins are regulating insulin secretion at physiological concentrations of glucose. The mechanisms by which kisspeptins regulate islet function and insulin secretion are unknown and will be further investigated.     

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.     

Long-term effect of Ph on B-cell function in isolated islets of langerhans in tissue culture

Summary Collagenase isolated mouse pancreatic islets were maintained in tissue culture for up to 5 months in a culture medium buffered with Hepes and the pH varying between 6.8 and 7.6. The amount of insulin released into the medium and the insulin response to glucose and glucose plus theophylline were measured during the culture period. It was found that islets cultured at pH 7.2 maintained the ability to release insulin into the medium for at least 5 months, which was longer than islets cultured at the other pH values. During the first weeks, the islets cultured at pH 7.6 had a higher response to both glucose and glucose plus theophylline than islets cultured at the other pH values, but later they lost their insulin releasing ability.     

Differential mechanisms of glucose and palmitate in augmentation of insulin secretion in mouse pancreatic islets

Aims/hypothesis. To assess the possible importance of saturated fatty acids in glucose amplification of K⁺ _ATP channel-independent insulin secretion. Methods. Insulin release from perifused pancreatic islets of NMRI mice was determined by radioimmunoassay. Results. In the presence of K⁺ (20 mmol/l) and diazoxide (250 μmol/l), which stimulates Ca²⁺ influx and opens K⁺ _ATP channels, palmitate (165 μmol/l total; 1.2 μmol/l free) increased insulin secretion at 3.3, 10 and 16.7 mmol/l glucose while glucose (10; 16.7 mmol/l) did not increase insulin secretion. In the presence of K⁺ (60 mmol/l) and diazoxide (250 μmol/l), glucose (10; 16.7 mmol/l) stimulation of K⁺ _ATP channel-independent insulin secretion increased, whereas the effectiveness of palmitate (165 μmol/l total; 1.2 μmol/l free) on insulin secretion at both 3.3, 10 or 16.7 mmol/l glucose was reduced. Palmitate thereby mimicked the stimulatory pattern of the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l), which also failed to increase insulin secretion at maximum depolarising concentrations of K⁺ (60 mmol/l). Furthermore, the protein kinase C inhibitor calphostin C (1 μmol/l), led to a complete suppression of the effects of both palmitate (165 μmol/l total; 1.2 μmol/l free) and myristate (165 μmol/l total; 2.4 μmol/l free) stimulation of glucose (16.7 mmol/l)-induced insulin secretion. Calphostin C (1 μmol/l), however, failed to affect insulin secretion induced by glucose (16.7 mmol/l). Conclusion/interpretation. These data suggest that glucose could increase insulin secretion independently of saturated fatty acids like palmitate and myristate, which amplify glucose-induced insulin secretion by activation of protein kinase C. [Diabetologia (2001) 44: 738–746] Received: 30 October 2001 and in revised form: 31 January 2001     

Direct effects of progesterone on rat islets of Langerhans in vivo and in tissue culture

Summary Progesterone and oestradiol did not alter rates of insulin secretion from isolated rat islets of Langerhans during a 60 min period of incubation in vitro. However, islets isolated from rats which had been injected daily for 15 days with progesterone (5 mg) and oestradiol (5 g) showed enhanced rates of insulin secretion in response to stimulation by 20 mmol/l glucose or 6 and 20 mmol/l glucose plus 5 mmol/l theophylline. Islets from rats which had been injected with the slow-releasing depot progesterone derivative, hydroxyprogesterone hexanoate, 3 times in 15 days, also showed enhanced rates of insulin release in the absence of any alteration in adenylate cyclase activity. In neither experiment could increased food intake, blood glucose levels or islet insulin content account for the observed changes. The possibility of a direct effect of progesterone on the secretory process was investigated in islets which had been cultured for 20 h with progesterone and oestradiol; these islets were then subjected to a variety of stimuli for secretion. They responded significantly more to glucose (6 or 20 mmol/l) in the presence of theophylline (5 mmol/l), while their insulin content was not significantly different from control islets cultured for a similar period. Islets cultured for 20 h in the presence of progesterone and oestradiol did not show any change in their adenylate cyclase activities. Similarly, direct addition of progesterone and oestradiol to islet homogenates did not alter the adenylate cyclase activity during a 30 minute incubation. These results suggest that progesterone and oestradiol affect insulin secretion directly, by a mechanism which does not involve activation of adenylate cyclase.     

Glutamate decarboxylase 67 contributes to compensatory insulin secretion in aged pancreatic islets

Pancreatic islets play an essential role in regulating blood glucose levels. Age-dependent development of glucose intolerance and insulin resistance results in hyperglycemia, which in turn stimulates insulin synthesis and secretion from aged islets, to fulfill the increased demand for insulin. However, the mechanism underlying enhanced insulin secretion remains unknown. Glutamic acid decarboxylase 67 (GAD67) catalyzes the conversion of glutamate into γ-aminobutyric acid (GABA) and CO₂. Both glutamate and GABA can affect islet function. Here, we investigated the role of GAD67 in insulin secretion in young (3 month old) and aged (24 month old) C57BL/6J male mice. Unlike young mice, aged mice displayed glucose-intolerance and insulin-resistance. However, aged mice secreted more insulin and showed lower fed blood glucose levels than young mice. GAD67 levels in primary islets increased with aging and in response to high glucose levels. Inhibition of GAD67 activity using a potent inhibitor of GAD, 3-mercaptopropionic acid, abrogated glucose-stimulated insulin secretion from a pancreatic β-cell line and from young and aged islets. Collectively, our results suggest that blood glucose levels regulate GAD67 expression, which contributes to β-cell responses to impaired glucose homeostasis caused by advanced aging.     

Total parenteral nutrition modulates hormone release by stimulating expression and activity of inducible nitric oxide synthase in rat pancreatic islets

The expression and activities of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) in relation to insulin and glucagon secretory mechanisms were investigated in islets isolated from rats subjected to total parenteral nutrition (TPN) for 10 d. TPN is known to result in significantly increased levels of plasma lipids during the infusion time. In comparison with islets from freely fed control rats, islets taken from TPN rats at d 10 displayed a marked decrease in glucose-stimulated insulin release (4.65±0.45 ng/[islet·h] vs 10.25±0.65 for controls) (p<0.001) accompanied by a strong iNOS activity (18.3±1.1 pmol of NO/[min·mg of protein]) and a modestly reduced cNOS activity (11.3±3.2 pmol of NO/[min.mg of protein] vs 17.7±1.7 for controls) (p<0.01). Similarly, Western blots showed the expression of iNOS protein as well as a significant reduction in cNOS protein in islets from TPN-treated rats. The enhanced NO production, which is known to inhibit glucose-stimulated insulin release, was manifested as a strong increase in the cyclic guanosine 5′-monophosphate content in the islets of TPN-treated rats (1586±40 amol/islet vs 695±64 [p<0.001] for controls). Moreover, the content of cyclic adenosine monophosphate (cAMP) was greatly increased in the TPN islets (80.4±2.1 fmol/islet vs 42.6±2.6 [p<0.001] for controls). The decrease in glucose-stimulated insulin release was associated with an increase in the activity of the secretory pathway regulated by the cAMP system in the islets of TPN-treated rats, since the release of insulin stimulated by the phosphodiesterase inhibitor isobutylmethylxanthine was greatly increased both in vivo after iv injection and after in vitro incubation of isolated islets. By contrast, the release of glucagon was clearly reduced in islets taken from TPN-treated rats (33.5±1.5 pg/[islet·h] vs 45.5±2.2 for controls) (p<0.01) when islets were incubated at low glucose (1.0 mmol/L). The data show that long-term TPN treatment in rats brings about impairment of glucose-stimulated insulin release, that might be explained by iNOS expression and a marked iNOS-derived NO production in the β-cells. The release of glucagon, on the other hand, is probably decreased by a direct “nutrient effect” of the enhanced plasma lipids. The results also suggest that the islets of TPN-treated rats have developed compensatory insulin secretory mechanisms by increasing the activity of their β-cell cAMP system.     

Regulation,perturbation, and correction of metabolic events in pancreatic islets

The physiological regulation of nutrient catabolism in islet cells, its perturbation in non-insulin-dependent diabetes mellitus, and the tools available to compensate for such a perturbation are reviewed. In terms of physiology, emphasis is placed on the relevance of glucokinase to hexose-induced insulin release, protein-to-protein interaction and enzyme-to-enzyme channelling, and the preferential stimulation of mitochondrial oxidative events in glucose-stimulated B-cells. In terms of pathology, attention is drawn to the deficiency of FAD-linked mitochondrial glycerophosphate dehydrogenase. Last, as far as therapeutic aspects are concerned, the potential usefulness of hypoglycemic sulfonylureas and meglitinide analogs, adenosine analogs, non-glucidic nutrients, and GLP-1 is underlined.Invited lecture presented during the 6th International Milano Meeting on Diabetes held in Milan on 21–23 March, 1996     

The dual effect of isoproterenol on insulin release is suppressed in pancreatic islets from hypothalamic obese rats

Hyperinsulinemia in obesity has been attributed to insulin oversecretion by pancreatic beta-cells. Beta-cells are equipped with cholinergic and adrenergic receptors; whereas overall acetylcholine action is to potentiate, catecholamines' effect is to inhibit glucoseinduced insulin release (GIIR) via α₂-adrenoreceptor. However, it has been shown that \-adrenergic agonists potentiate glucose response. GIIR was studied in pancreatic islets from hyperinsulinemic adult obese rats, obtained by l-glutamate monosodium (MSG) neonatal treatment. Islets from MSG-rats were more glucose responsive than control ones. Isoproterenol, a \-adrenergic agonist, inhibited the GIIR in islets from MSG-obese rats. Results indicate that MSG treatment causes alteration on function of beta-cell adrenoceptors.