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.     

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.     

The effect of monooleoylglycerol on insulin secretion from isolated perifused rat islets

Summary The effect of monooleoylglycerol on cholecystokinin-and tolbutamide-induced insulin secretion was examined in isolated perifused rat islets. In the presence of 5.5 mmol/l glucose, addition of 10 nmol/l cholecystokinin or 50 mol/l tolbutamide had practically no effect on insulin secretion. Combined tolbutamide and cholecystokinin led to a biphasic insulin secretory response which was significantly enhanced by addition of 50 mol/l monooleoylglycerol, an inhibitor of diacylglycerol kinase. Monooleoylglycerol (50 mol/l) alone had a minimal stimulatory effect on insulin release in the presence of 5.5 mmol/l glucose. Perifusion of islets with 1 mol/l forskolin had no significant effect on basal insulin secretion in the presence of 5.5 mmol/l glucose, but markedly enhanced the responses to both cholecystokinin plus tolbutamide, and to the combination of cholecystokinin, tolbutamide and monooleoylglycerol. Lowering the glucose level to 2.75 mmol/l abolished the profound stimulatory effect to these agonist combinations on insulin release. Finally, monooleoylglycerol also enhanced the first and second phase insulin secretory responses induced by 20 mmol/l glucose. These results are discussed in relationship to the possible role of protein kinase C in mediating insulin secretion.     

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.     

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.     

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.     

Leptin inhibition of insulin secretion from isolated human islets

Leptin is a hormone produced and secreted from the adipose tissue. Its physiological actions include the regulation of satiety, food intake and energy balance. The production of leptin is increased by high insulin levels. Here, we demonstrate that leptin acts as an inhibitor of glucose-induced (20 mM) insulin secretion from isolated human islets. No effect was observed in the presence of lower glucose levels (2.8 and 10 mM glucose). The pancreatic β-cell might represent a target of a direct physiological action of leptin. We suggest the presence of an “adipo-insular axis” in which leptin mediates negative feedback from the adipose tissue to the endocrine pancreas. Received: 21 July 1997 / Accepted in revised form: 1 October 1997     

Effect of enkephalins and morphine on insulin secretion from isolated rat islets

Summary The direct effects of an enkephalin analogue, (D-Ala²/MePhe⁴/Met/(O)-ol) enkephalin (DAMME), on insulin release from isolated islets of Langerhans of the rat have been investigated. DAMME had a dose-dependent effect on insulin secretion: low concentrations (10^–10 to 10^–8 mol/l) were stimulatory while high concentrations (10^–5mol/l) were inhibitory in the presence of 8 mmol/l glucose. Similar effects were found with met-enkephalin, and with the longer acting alanine substituted metenkephalin. Morphine sulphate (5 sx 10^–7 mol/l) also stimulated insulin release. The effects of enkephalin and morphine were blocked by the specific opiate antagonist naloxone hydrochloride (1.2 × 10^–6 mol/l). The insulin secretory response of perifused islets to enkephalins and morphine was rapid, corresponding to the first phase of glucose induced insulin release. These observations suggest that there may be opiate receptors in islets, and that opioid peptides could modulate insulin release.     

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.     

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.     

Investigations on isolated islets in vitro. IX. Influence of food deprivation and glucose refeeding in vitro on insulin secretion

Summary Starvation resulted in a progressive decrease of insulin secretion in response to 15 mM glucose, but it neither inhibited insulin release completely nor changed the shape of the glucose response curve. Long-term fasting caused the glucose threshold to rise to 30 mM glucose during an incubation time of 15 min, whereas fed animals were characterized by a sigmoidal insulin response and a threshold level of 5 mM glucose during short-term incubation, too. Prolongation of incubation time (60 or 120 min) or pretreatment of isletsin vitro with 20 mM glucose for 60 min caused progressive lowering of the glucose threshold, indicating that glucoseper se influenced the effective stimulating concentration. The effect of starvation is not specific for glucose, since hormone secretion in response to glibenclamide, nicotinic acid and theophylline was also reduced, whereas theophylline can partly counteract the diminished release. Although the content of insulin as well as glucagon in the islets was reduced by food deprivation it is assumed that the islet hormone content is not responsible for the rise of the glucose threshold caused by starvation.     

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.     

Correction of diabetic pattern of insulin release from islets of the spiny mouse (Acomys cahirinus) by glucose priming in vitro

Summary Insulin release kinetics were studied in perifused islets of Langerhans, isolated from mildly hyperglycaemic and from normoglycaemic spiny mice (Acomys cahirinus), a rodent predisposed to develop spontaneously non-ketotic diabetes. In both groups, insulin response to glucose (16.7 mmol/l) was delayed in comparison with that of rat islets, the release kinetics being analogous to that of human Type 2 (non-insulin-dependent) diabetes. Thirty min priming of the isolated Acomys islets with glucose (16.7 mmol/l) resulted in potentiation of the insulin release to a second stimulation. The degree of potentiation decreased exponentially with the time interval between stimulations, showing a t 1/2 of 18 min. Induction of potentiation by glucose was time-dependent, giving a maximal effect after 20 min of priming. In addition to overall amplification of the insulin response, priming with glucose accelerated markedly the initial release rates, correcting the dynamics of the response. We conclude that: (1) decreased and delayed insulin secretion is found in Acomys cahirinus before the development of hyperglycaemia; (2) induction of time-dependent potentiation in the islet by priming with glucose corrects the diabetic-type dynamics of insulin release; (3) therefore the deficient insulin release of Acomys is of a functional nature, the mechanism of potentiation bypassing the defect; (4) since insulin release in Acomys resembles that in prediabetic and diabetic man, similar conclusions might apply to the islet dysfunction in Type 2 diabetes.     

Influence of isolated insulin antibodies on the insulin secretion of the islets of Langerhans in vitro

Summary Mouse islets of Langerhans, isolated by microdissection after treatment with collagenase, were incubated either with pure insulin antibodies (IAB), which were prepared by immune precipitation, or with exogenous insulin. Insulin release was enhanced with increased concentrations of IAB and was inhibited by exogenous insulin. The results suggest that it was not the insulin per se, but probably its biological effect on the -cells that influenced insulin secretion.     

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 extracellular pH on insulin secretion and glucose metabolism in neonatal and adult rat pancreatic islets

Changes in extracellular pH are known to affect glucose-stimulated insulin secretion. In the present study, glucose metabolism in pancreatic islets cultured at different pHs was investigated. Also, for islet transplantation purposes, insulin secretion and glucose metabolism were compared in neonatal and adult islets at different pHs to determine which islet preparation is more tolerant to acidity and alkalinity. The results revealed a dependency of insulin secretion on the external pH in both neonatal and adult islets. Reduction of insulin secretion was observed at both the acidic and alkaline sides of pH 7.3. Glucose stimulated increases of insulin secretion in all cases. Similar results were obtained for ATP and pyruvate contents. Intracellular insulin increased with the increase of pH value. In contrast, calcium content decreased with the increase of pH. The results demonstrate that neonatal islets are more acid tolerant than adult islets. Both basal and glucose-stimulated insulin secretions, as well as other parameters of neonatal islets were significantly higher than those of adult islets in response to low pH. The differences under alkaline conditions were not significant but give an indication that neonatal islets are more tolerant to alkalinity than are adult islets. Received: 10 February 2001 / Accepted in revised form: 29 June 2001     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

分离SD大鼠胰岛接种于24孔板中,用不同浓度的葡萄糖和白藜芦醇分别培养1 h或24 h,结果表明白藜芦醇孵育大鼠胰岛1 h可呈剂苗依赖地抑制大鼠高糖刺激的胰岛素分泌,1、10和100 μmol/L白藜芦醇可以分别使胰岛素的分泌降低10%、35%(P<0.05)和80%(P<0.01).显微离子成像技术爪10μmol/L的白藜芦醇可以使高糖引起的β细胞内Ca2+浓度的升高减少60%(P<0.05).白藜芦醇可使软脂酸孵育24 h大鼠胰岛的胰岛素分泌恢复到对照组的75%(P<0.01),提示白藜芦醇短期可通过调控细胞内的Ca+浓度,而抑制原代胰岛高精刺激的胰岛素分泌,长期可改善软脂酸引起的β细胞损伤.     

Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans

Aims: The role of cannabinoid receptors in human islets of Langerhans has not been investigated in any detail, so the current study examined CB1 and CB2 receptor expression by human islets and the effects of pharmacological cannabinoid receptor agonists and antagonists on insulin secretion. Methods: Human islets were isolated from pancreases retrieved from heart‐beating organ donors. Messenger RNAs encoding human CB1 and CB2 receptors were amplified from human islet RNA by RT‐PCR and receptor localization within islets was identified by immunohistochemistry. Dynamic insulin secretion from human islets perifused with buffers supplemented with CB1 and CB2 receptor agonists and antagonists was quantified by radioimmunoassay. Results: RT‐PCR showed that both CB1 and CB2 receptors are expressed by human islets and immunohistochemistry indicated that receptor expression co‐localized with insulin‐expressing β‐cells. Perifusion experiments using isolated human islets showed that insulin secretion was reversibly stimulated by both CB1 and CB2 receptor agonists, with CB1 receptor activation associated with increased basal secretion whereas CB2 receptors were coupled to initiation and potentiation of insulin secretion. Antagonists at CB1 (N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) and CB2 (N‐(1,3‐Benzodioxol‐5‐ylmethyl)‐1,2‐dihydro‐7‐methoxy‐2‐oxo‐8‐(pentyloxy)‐3‐quinoline carboxamide) receptors failed to inhibit the stimulatory effects of the respective agonists and, unexpectedly, reversibly stimulated insulin secretion. Conclusions: These data confirm the expression of CB1 and CB2 receptors by human islets and indicate that both receptor subtypes are coupled to the stimulation of insulin secretion. They also implicate involvement of CB1/2 receptor‐independent pathways in the antagonist‐induced stimulatory effects.     

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.     

Insulin,glucagon and somatostatin secretion by cultured islets from normal and diabetic hamsters

Summary The interhormonal relationship within the pancreatic islets have been studied by previous investigators, but the cellular interplay and the sequence of events in the islet cell's response to stimulators has remained unclear. In the present study, pancreatic islets were isolated by collagenase digestion from normal and streptozotocin-diabetic hamsters the latter being maintained with insulin treatment. The diabetic animals were used to provide A- and B-cell enriched islets. The islets from normal and diabetic hamsters were cultured in medium 199 plus 10% fetal calf serum with 0.8 or 5 mg/ml glucose. The cultures were maintained for up to seven days