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.     

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.     

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.     

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.     

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.     

Increased somatostatin secretion from pancreatic islets of streptozotocin-diabetic rats in response to glucose

Glucose stimulates somatostatin release from perifused pancreatic islets of diabetic rats 42–47 days after the induction of diabetes, and 48 h after withdrawal of insulin replacement therapy.

The glucose effect is augmented by theophylline or glucagon.

Basal somatostatin release and glucose-induced secretion are significantly higher in diabetic islets than in controls.

It is suggested that glucose promotes somatostatin release by directly interacting with islet D cells but not via indirect pathways. Glucose-induced stimulation appears to be modulated by a D-cell adenylate cyclase/phosphodiesterase system. Reasons responsible for increased somatostatin secretion by diabetic islets include reduction in B-cell mass, suggesting that B cells may normally suppress the secretory activity of D cells.     

Effect of amino-acids on secretion of insulin by isolated islets of the monkey

Summary Insulin secretion was monitored in monkey islets isolated by collagenase digestion and exposed to leucine and arginine with and without glucose. Leucine by itself (10 to 40 mmol/l) elicited concentration-dependent insulin secretion. At 40 mmol/l, leucine was approximately 60% as effective as glucose (16.7 mmol/l). The response to leucine was increased at low glucose concentrations. In high concentrations (20 and 40 mmol/l), arginine by itself was a poor stimulant. The effect of arginine was enhanced at low glucose concentrations (2.8 to 5.6 mmol/l). At high glucose concentrations neither amino-acid produced any significant further increase in insulin release.     

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.     

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.     

The priming effect of glucose on insulin secretion from isolated islets of Langerhans

Summary Biphasic insulin secretion from perifused rat islets of Langerhans was enhanced if islets had previously been stimulated with glucose 16.6 mmol/l. The priming effect of glucose was reduced if mannoheptulose (16.6 mmol/l), deuterium oxide (D₂O; 98% v/v) or adrenaline (10mol/l) was included in the medium during the initial stimulation period, or if Calcium was omitted. Glyceraldehyde (16.6 mmol/l) but not theophylline (5 mmol/l) could substitute for glucose during the initial stimulation and make islets more responsive to subsequent stimulation. The results suggest that the priming effect of glucose on insulin secretion may be related to 1) glucose metabolism and 2) Ca fluxes in the B cell and the consequent activation of the microtubular system. Neither the generation of intracellular cyclic AMP nor the release of insulin per se appears to be involved in the priming process.     

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.     

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.     

Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets

Summary Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied in an attempt to optimize the maintenance of glucose stimulated insulin biosynthesis and release in the cultured specimens. Islets which had been cultured at a physiological glucose concentration (5.5 mmol/l) in the absence of serum had an impaired glucose-stimulated insulin biosynthesis and release as well as a reduced insulin content. Thus, insulin biosynthesis was three times higher after culture in a serum supplemented medium. Further, the insulin secretion of islets cultured in the presence of serum was markedly enhanced in acute incubations with high concentrations of glucose. This response was most pronounced in islets which had been cultured free-floating. A comparison between different culture media showed that islets cultured in RPMI 1640 had the highest insulin production. The present data suggest that the most favourable conditions for long-term storage of isolated islets in culture may be obtained when the islets are maintained as free-floating explants in a culture medium consisting of RPMI 1640 supplemented with serum.     

Young capillary vessels rejuvenate aged pancreatic islets

Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature.Aging leads to progressive decline of various homeostatic processes in mammals, including a deteriorating regulation of blood glucose levels. Pancreatic islets are small organs composed of endocrine cells that secrete the major hormones insulin, glucagon, and somatostatin, which play a key role in regulating blood glucose levels. Age-dependent dysfunction of islets and the concomitant dysregulation of blood glucose levels increase the risk for type 2 diabetes (1), which in turn contributes to other age-related chronic diseases. In general, it has been assumed that aging causes an intrinsic dysfunction of the insulin-secreting beta cells through reduced proliferative capacity and/or defective insulin secretion (1–9). However, there have been numerous reports that age-dependent impairment of glucose homeostasis is not just a result of intrinsic, age-dependent dysfunction of islets but is also caused by systemic factors. For example, islet function may be compromised by age-related increases in adiposity (10, 11) and by bloodborne factors (12), or it could be affected indirectly by age-related deficiencies in vascular remodeling (13). Thus, the replicative decline of old pancreatic beta cells can be attributed to systemic factors (12). Recent studies identified factors present in young blood that reverse age-related cognitive impairments and induce vascular remodeling and regeneration in the brain and skeletal muscle (14–16), but so far it has not been feasible to discriminate systemic influences from aging factors intrinsic to islet endocrine cells. Here we address the long-standing question of whether the age-dependent impairment of glucose homeostasis is caused by intrinsic, age-dependent dysfunction of islets or by systemic aging factors.Our strategy to discern age-related intrinsic changes in islet function was to study islets from young mature (2 mo) and aged (18 mo) mice and to follow these same groups of islets in three different environments: in vivo in the body of young and aged mice, in vitro after isolation, and again in vivo after transplantation into the anterior chamber of the eye in young mice (17). We also examined a large number of human islets from young mature and old pancreatic donors (17–65 y of age). We hypothesized that islets are affected by the systemic milieu, such that the effects the aged organism exerts on the islet can be rescued in a young organism. We characterized islet structure and function at the molecular, anatomic, and physiologic levels to distinguish intrinsic from systemic factors impinging on the islet as the organism ages. Our results reveal that aging of islets involves little intrinsic decline of beta cell function but is accompanied by malfunctioning blood vessels, suggesting that age-impaired glucose homeostasis is not caused by the intrinsic aging of beta cells but, rather, is a result of vascular aging that can be reversed by placing aged islets in a young environment.     

The atypical antipsychotic clozapine impairs insulin secretion by inhibiting glucose metabolism and distal steps in rat pancreatic islets

Aims/hypothesis Diabetogenic effects of some atypical antipsychotic drugs have been reported, although the mechanisms are not fully understood. We investigated the long-term effects of culturing isolated rat pancreatic islets with atypical antipsychotic clozapine.Methods Glucose- and non-glucose-stimulated insulin secretion, glucose metabolism and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured in islets cultured with or without clozapine.Results Although acute incubation or 3-day culture with clozapine did not affect glucose-stimulated insulin secretion, clozapine suppressed glucose-stimulated insulin secretion by 53.2% at 1.0 μmol/l (therapeutic concentration) after 7 days of culture. Islet glucose oxidation and [Ca²⁺]_i elevation by high glucose were not affected after 3 days of culture, but clozapine significantly inhibited islet glucose oxidation, ATP production, and [Ca²⁺]_i elevation by high glucose after 7 days of culture. Moreover, 7 days of culture with clozapine inhibited insulin secretion stimulated by: (1) membrane depolarisation induced by high K⁺; (2) protein kinase C activation; and (3) mastoparan at 16.7 mmol/l glucose under stringent Ca²⁺-free conditions. Elevation of [Ca²⁺]_i by high K⁺-induced membrane depolarisation was similar in control and clozapine-treated islets. Clozapine, a muscarinic blocker, acutely inhibited carbachol-induced insulin secretion, as did atropine, whereas after 7 days of culture atropine did not have the inhibitory effect shown by clozapine after 7 days. The impairment of glucose-stimulated insulin secretion recovered 3 days after the removal of clozapine treatment.Conclusions/interpretation The present study demonstrated that the atypical antipsychotic drug clozapine directly impaired insulin secretion via multiple sites including glucose metabolism and the distal step in insulin exocytosis in a long-term culture condition. These mechanisms may be involved in the form of diabetes mellitus associated with atypical antipsychotic drugs.     

Mechanisms of octanoic acid potentiation of insulin secretion in isolated islets

ABSTRACT

A potentiating effect of medium-chain triglycerides on glucose-stimulated insulin secretion (GSIS) has been observed since the 1960s. Subsequent observations identified octanoic acid (OA), the main component of medium-chain triglyceride, as the potentiator of GSIS, but the mechanism was unclear. We used wild-type (WT), short-chain 3-hydroxyacyl-CoA dehydrogenase knockout (Hadh^-/-), and sulfonylurea receptor 1 knockout (Sur1^-/-) mouse islets to define the mechanism of OA potentiation of insulin secretion. Application of OA alone induced a 2- to 3- fold increase of insulin secretion with an apparent threshold of 3 mM in WT mouse islets, suggesting that OA itself is a weak insulin secretagogue. However, OA at 1 mM strongly potentiated fuel-stimulated insulin secretion, especially GSIS. The potentiating effect on fuel-stimulated insulin secretion by OA did not require fatty acid β-oxidation because OA also potentiated amino acid-stimulated insulin secretion in islets isolated from Hadh^-/- mice, which cannot fully oxidize OA. Measurements using Sur1^-/- islets indicated that the potentiating effect of OA on fuel-stimulated insulin secretion is Ca²⁺ dependent and is often accompanied by β-cell membrane potential depolarization, and may also involve the Ca²⁺/calmodulin complex. Experiments using DCPIB, an ethacrynic acid derivative, to inhibit volume-sensitive anion channels (VSACs) in Sur1^-/- islets demonstrated that the potentiation effects of OA on insulin secretion are in part medicated by activation of VSAC. In addition, inhibition of IP3 receptor also abolishes the OA-induced intracellular Ca²⁺ increase in Sur1^-/- islets.     

Immaturity of insulin secretion by pancreatic islets isolated from one human neonate

Human β‐cells are functionally mature by the age of 1 year. The timeline and mechanisms of this maturation are unknown owing to the exceptional availability of testable tissue. Here, we report the first in vitro study of insulin secretion by islets from a 5‐day‐old newborn. Glucose was inefficient alone, but induced insulin secretion, which was concentration‐dependent, showed a biphasic time‐course and was of similar magnitude as in infant islets when β‐cell cyclic adenosine monophosphate was raised by forskolin. Tolbutamide alone was effective in low glucose, but its effect was not augmented by high glucose. Metabolic amplification by glucose was thus inoperative, in contrast to amplification by cyclic adenosine monophosphate. Newborn islets showed high basal insulin secretion that could be inhibited by diazoxide or omission of CaCl₂. Postnatal acquisition of functional maturity by human β‐cells implicates control of basal secretion and production of metabolic signals able to activate both triggering and amplifying pathways of insulin secretion.