Comparative effects of amino acids and glucose on insulin secretion from isolated rat or mouse islets

Glucose and the combination of leucine and glutamine were used to stimulate insulin secretion from rat islets during a dynamic perifusion and the responses obtained were compared with those elicited from mouse islets under identical conditions. In rat islets, glucose (15 mM) or the amino acid combination of 10 mM glutamine plus 20 mM leucine were most efficacious and peak second-phase insulin release responses were 20- to 30-fold above prestimulatory rates. In contrast to rat islet responses, sustained second-phase insulin secretory responses to the same agonists were minimally increased 1- to 2-fold from mouse islets. Parallel studies demonstrated that phospholipase C (PLC) was markedly activated in rat, but not mouse, islets by both high glucose concentrations and the amino acid combination. Additional studies documented that glucose and amino acid responses of both rat and mouse islets were amplified by carbachol or forskolin. However, wortmannin, a phosphatidylinositol 3-kinase inhibitor, amplified only the responses to glucose leaving the responses to the amino acid mixture unaltered. These observations support the concept that mitochondrial metabolism alone is minimally effective in stimulating insulin secretion from islets. The activation of the supplementary second messenger systems (PLC and/or cAMP) appears essential for the emergence of their full secretory potential. The mechanism regulating the potency and specificity of wortmannin's impact on glucose-induced secretion remains to be identified; however a unique mechanism is supported by these findings.     

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.     

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 permissive effect of glucose,tolbutamide and high K+ on arginine stimulation of insulin release in isolated mouse islets

Summary Mouse islets were used to study how glucose modulates arginine stimulation of insulin release. At 3 mmol/l glucose, arginine (20 mmol/l) decreased the resting membrane potential of B cells by about 10 mV, but did not evoke electrical activity. This depolarisation was accompanied by a slight but rapid acceleration of ⁸⁶Rb⁺ efflux and ⁴⁵Ca²⁺ influx. However, ⁴⁵Ca²⁺ efflux and insulin release increased only weakly and belatedly. When the membrane was depolarised by threshold (7 mmol/l) or stimulatory (10–15 mmol/l) concentrations of glucose, arginine rapidly induced or augmented electrical activity, markedly accelerated ⁸⁶Rb⁺ efflux, ⁴⁵Ca²⁺ influx and efflux, and triggered a strong and fast increase in insulin release. When glucose-induced depolarisation of the B-cell membrane was prevented by diazoxide, arginine lost all effects but those produced at low glucose. However, the delayed increase in release still exhibited some glucose-dependency. In contrast, depolarisation by tolbut amide, at low glucose, largely mimicked the permissive effect of high glucose. Depolarisation by high K⁺ also amplified arginine stimulation of insulin release, but did not accelerate it as did glucose or tolbutamide. Omission of extracellular Ca²⁺ abolished the releasing effect of arginine under all conditions. The results thus show that the permissive action of glucose mainly results from its ability to depolarise the B-cell membrane. It enables the small depolarisation by arginine itself to activate Ca channels more rapidly and efficiently. Changes in the metabolic state of B cells may also contribute to this permissive action by increasing the efficacy of the initiating signal triggered by arginine.     

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     

Divergent effects of epinephrine and prostaglandin E2 on glucose-induced insulin secretion from perifused rat islets

The impact of the catecholamine epinephrine and the postulated inhibitory second messenger prostaglandin E(2) (PGE(2)) on the kinetics and magnitude of glucose-induced insulin secretion were compared and contrasted. In agreement with a number of studies, epinephrine was a most effective antagonist of glucose-induced insulin secretion. Dose-response studies using 8 to 10 mmol/L glucose as stimulant established that levels as low as 1 to 10 nmol/L of the catecholamine were effective at inhibiting release. Glucose (20 mmol/L) caused an approximately 25-fold increase in insulin secretion, an effect that was completely abolished by 1 micromol/L epinephrine. Under conditions where it completely abolished 20 mmol/L glucose-induced insulin release, epinephrine (1 micromol/L) reduced, but did not abolish, the stimulatory effect of glucose on phospholipase C activation. Chronic 3-hour exposure to 10 mmol/L glucose alone desensitized the islet to subsequent stimulation by glucose. Despite its ability to completely suppress secretion to 10 mmol/L glucose, epinephrine failed to protect the islet from hyperglycemia-induced desensitization. In sharp contrast to epinephrine, PGE(2) at levels ranging from 1 to 10 micromol/L had no discernible adverse effect on 10 mmol/L glucose-induced secretion. These findings suggest that multiple mechanisms contribute to the inhibitory impact of epinephrine on release and, in conjunction with other studies, cast serious doubt on the concept that PGE(2) plays any significant inhibitory role in the regulation of glucose-induced secretion.     

Galanin fragments and analogues: effects on glucose-stimulated insulin secretion from isolated rat islets.

Galanin occurs in intrapancreatic nerves and inhibits insulin secretion both in vivo and in vitro. To investigate which part of the galanin molecule accounts for this inhibition, we studied the effect of porcine galanin, four galanin fragments and three galanin analogues with substitutions of the 2nd amino acid in galanin, on glucose-stimulated insulin secretion from isolated rat islets cultured overnight. The islets were incubated for 1 h at 8.3 mM glucose. Porcine galanin1-29 inhibited insulin secretion at dose levels from 10(-8) M to 10(-6) M (p less than 0.001). Also, the galanin fragments GAL1-15, GAL2-29, and GAL3-29 significantly inhibited insulin secretion at and above concentrations of 10(-7) M (p less than 0.001), 10(-8) M (p less than 0.001), and 10(-7) M (p less than 0.001), respectively. Galanin analogues where the 2nd N-terminal amino acid had been changed from tryptophan to tyrosine (GAL-TYR2) or isoleucine (GAL-ILE2) inhibited insulin secretion, as did porcine galanin1-29, whereas after substitution with phenylalanine (GAL-PHE2) no effect was observed. Furthermore, the C-terminal fragment GAL10-29 did not influence insulin secretion. We conclude that the inhibitory action by galanin on glucose-stimulated insulin secretion from normal islets resides in the N-terminal part of the molecule. In contrast, the C-terminal part of galanin apparently does not influence insulin secretion.     

Calcineurin increases glucose activation of ERK1/2 by reversing negative feedback

In pancreatic β cells, ERK1 and ERK2 participate in nutrient sensing, and their activities rise and fall as a function of glucose concentration over the physiologic range. Glucose metabolism triggers calcium influx and release of calcium from intracellular stores to activate ERK1/2. Calcium influx also activates the calcium-dependent phosphatase calcineurin, which is required for maximal ERK1/2 activation by glucose. Calcineurin controls insulin gene expression by ERK1/2-dependent and -independent mechanisms. Here, we show that, in β cells, glucose activates the ERK1/2 cascade primarily through B-Raf. Glucose activation of B-Raf, like that of ERK1/2, is calcineurin-sensitive. Calcineurin binds to B-Raf in both unstimulated and stimulated cells. We show that B-Raf is a calcineurin substrate; among calcineurin target residues on B-Raf is T401, a site of negative feedback phosphorylation by ERK1/2. Blocking calcineurin activity in β cells prevents dephosphorylation of B-Raf T401 and decreases B-Raf and ERK1/2 activities. We conclude that the major calcineurin-dependent event in glucose sensing by ERK1/2 is the activation of B-Raf.     

高浓度葡萄糖对PDX-1表达和胰岛素分泌功能的影响

目的 探讨高浓度葡萄糖（Glu）对PDX-1表达的影响及其与胰岛素（Ins）分泌的关系。方法 分别测定SD大鼠胰岛细胞基础和Glu刺激后Ins分泌量、细胞内Ins含量、细胞内PDX-1 mRNA和蛋白的表达水平。结果 1．高糖刺激3天后，基础和Glu刺激后Ins分泌量、细胞内Ins含量及PDX-1蛋白表达水平均明显降低（P〈0．01）。2．在高糖环境下，延长培养时间可显著加强高糖对PDX-1蛋白表达的抑制作用。3．纠正高糖环境3天后可部分逆转高糖对PDX-1蛋白表达的抑制作用。结论 高浓度Glu对PDX-1蛋白表达的抑制是Glu毒性作用的机制之一，纠正高糖3天后可部分逆转高糖对PDX-1蛋白表达的抑制作用，恢复Ins分泌功能。     

G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1

Aims/hypothesis  

Activation of the G protein-coupled receptor (GPR)40 by long-chain fatty acids potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, and GPR40 agonists are in clinical development for type 2 diabetes therapy. GPR40 couples to the G protein subunit Gα_q/11 but the signalling cascade activated downstream is unknown. This study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids.     

Doc2b enrichment enhances glucose homeostasis in mice via potentiation of insulin secretion and peripheral insulin sensitivity

Aims/hypothesis

Insulin secretion from pancreatic beta cells and insulin-stimulated glucose uptake into skeletal muscle are processes regulated by similar isoforms of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) and mammalian homologue of unc-18 (Munc18) protein families. Double C2 domain β (Doc2b), a SNARE- and Munc18-interacting protein, is implicated as a crucial effector of glycaemic control. However, whether Doc2b is naturally limiting for these processes, and whether Doc2b enrichment might exert a beneficial effect upon glycaemia in vivo, remains undetermined.

Methods

Tetracycline-repressible transgenic (Tg) mice engineered to overexpress Doc2b simultaneously in the pancreas, skeletal muscle and adipose tissues were compared with wild-type (Wt) littermate mice regarding glucose and insulin tolerance, islet function in vivo and ex vivo, and skeletal muscle GLUT4 accumulation in transverse tubule/sarcolemmal surface membranes. SNARE complex formation was further assessed using Doc2b overexpressing L6-GLUT4-myc myoblasts to derive mechanisms relatable to physiological in vivo analyses.

Results

Doc2b Tg mice cleared glucose substantially faster than Wt mice, correlated with enhancements in both phases of insulin secretion and peripheral insulin sensitivity. Heightened peripheral insulin sensitivity correlated with elevated insulin-stimulated GLUT4 vesicle accumulation in cell surface membranes of Doc2b Tg mouse skeletal muscle. Mechanistic studies demonstrated Doc2b enrichment to enhance syntaxin-4–SNARE complex formation in skeletal muscle cells.

Conclusions/interpretation

Doc2b is a limiting factor in SNARE exocytosis events pertinent to glycaemic regulation in vivo. Doc2b enrichment may provide a novel means to simultaneously boost islet and skeletal muscle function in vivo in the treatment and/or prevention of diabetes.     

Differential effects of beta-adrenergic agonists on insulin secretion from pancreatic islets isolated from rat and man

The selective beta 2-adrenergic agonist clenbuterol was ineffective as a stimulus for insulin secretion when isolated rat pancreatic islets were incubated with glucose at concentrations between 4 and 20 mM. Inclusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine led to potentiation of glucose-induced insulin secretion, but did not facilitate stimulation by clenbuterol. Furthermore, maintenance of isolated rat islets for up to 3 days in tissue culture also failed to result in the appearance of a secretory response to beta-agonists. By contrast, clenbuterol induced a dose-dependent increase in insulin release from isolated human islets incubated with 20 mM glucose. Clenbuterol did not increase the basal rate of insulin secretion (4 mM glucose) in human islets. Under perifusion conditions, the secretory response of human islets to clenbuterol was rapid, of similar magnitude to that seen under static incubation conditions and could be sustained for at least 30 min. The increase in insulin secretion induced by clenbuterol was inhibited by propranolol, indicating that the response was mediated by activation of beta-receptors. In support of this, a similar enhancement of glucose-induced insulin secretion was elicited by a different beta 2-agonist, salbutamol, in human islets. The results indicate that the B cells of isolated rat islets are unresponsive to beta-agonists, whereas those of human islets are equipped with functional beta-receptors which can directly influence the rate of insulin secretion.     

Impairment of the priming effect of glucose on insulin secretion from isolated islets of aging rats

The time-dependent potentiation (TDP) of insulin release or priming effect exerted by glucose was evaluated in the islets of Langerhans of mature and old rats. Islets isolated from 12-and 26-month-old male Sprague-Dawley rats and incubated for two consecutive 60-min periods in the presence of various stimulating agents were unable to enhance their insulin responsiveness significantly during the second incubation period and showed other abnormalities in their sensitivity to secretagogues compared with islets from 3-month-old animals. The priming action of glucose plus arginine or isobutylmethylxanthine (IBMX) was not observed in islets from 12-month-old rats, but surprisingly, islets from senescent rats showed a restoration of the beta-cell memory in the presence of IBMX. Interestingly, the islets isolated from 2-month-old animals previously exposed to an intravenous glucose load in vivo released approximately twice as much insulin as the islets taken from fed rats not subjected to the load. This potentiation exerted by the intravenous glucose administration was reduced but not abolished in the islets of glucose-intolerant, 12-month-old rats. In conclusion, the glucose TDP of insulin secretion is impaired in islets of mature and old rats, confirming an early loss of sensitivity of beta-cells to secretagogues during aging.     

Acute effects of fatty acids on insulin secretion from rat and human islets of Langerhans

Fatty acids have both stimulatory and inhibitory effects on insulin secretion. Long-term exposure to fatty acids results in impaired insulin secretion whilst acute exposure has generally been found to enhance insulin release. However, there are conflicting data in the literature as to the relative efficacy of various fatty acids and on the glucose dependency of the stimulatory effect. Moreover, there is little information on the responses of human islets in vitro to fatty acids. We have therefore studied the acute effects of a range of fatty acids on insulin secretion from rat and human islets of Langerhans at different glucose concentrations. Fatty acids (0.5 mM) acutely stimulated insulin release from rat islets of Langerhans in static incubations in a glucose-dependent manner. The greatest effect was seen at high glucose concentration (16.7 mM) and little or no response was elicited at 3.3 or 8.7 mM glucose. Long-chain fatty acids (palmitate and stearate) were more effective than medium-chain (octanoate). Saturated fatty acids (palmitate, stearate) were more effective than unsaturated (palmitoleate, linoleate, elaidate). Stimulation of insulin secretion by fatty acids was also studied in perifused rat islets. No effects were observed at 3.3 mM glucose but fatty acids markedly potentiated the effect of 16.7 mM glucose. The combination of fatty acid plus glucose was less effective when islets had been first challenged with glucose alone. The insulin secretory responses to fatty acids of human islets in static incubations were similar to those of rat islets. In order to examine whether the responses to glucose and to fatty acids could be varied independently we used an animal model in which lactating rats are fed a low-protein diet during early lactation. Islets from rats whose mothers had been malnourished during lactation were still able to respond effectively to fatty acids despite a lowered secretory response to glucose. These data emphasise the complex interrelationships between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis during undernutrition.     

Beneficial effects of lipids and prolactin on insulin secretion and beta-cell proliferation: a role for lipids in the adaptation of islets to pregnancy

To meet the increased demand for insulin during pregnancy, the pancreatic islets undergo adaptive changes including enhanced insulin secretion and beta-cell proliferation. These changes peak in mid-pregnancy and return to control levels by parturition. Because lactogens (placental lactogen and/or prolactin) induce this up-regulation and remain elevated throughout gestation, we examined whether lipids alter the effects of prolactin on islets. In response to prolactin, there was a 2.5-fold increase in insulin secretion when compared with control islets. There was also a 2.5-fold increase in insulin secretion in response to palmitate and a fivefold increase when islets were cultured with a combination of prolactin and palmitate. After culture with prolactin and palmitate, acute stimulation with 10 mM glucose for 1 h showed a suppression of insulin release. However, including palmitate in the stimulation media (a condition similar to late pregnancy in vivo) restored a higher rate of insulin release. This suggests that elevated lipids in late pregnancy lead to enhanced insulin secretion that is increasingly dependent on lipids and less sensitive to glucose. beta-Cell proliferation was also increased sixfold by prolactin and threefold with palmitate. The combination of both was slightly more than additive (11-fold). Similar experiments with oleate had no effect on insulin secretion. However, oleate stimulated beta-cell division by threefold and was synergistic with prolactin (21-fold). These results were repeated in experiments including normal serum. Interestingly, prolactin also blocked the reduction of glucokinase levels observed with fatty acids. Overall, these results suggest that increased lipids during pregnancy likely contribute to the adaptation of islets to pregnancy by further enhancing beta-cell division. In addition, the increase in lipids leads to enhanced insulin secretion that is less sensitive to glucose and more dependent on lipids. This provides a potential mechanism for maintaining elevated insulin secretion until parturition while preparing islets for normal glucose sensitivity post partum.     

The effect of epinephrine and isoproterenol on insulin secretion and glucose utilization in isolated islets of Langerhans from mice.

The effect of epinephrine and isoproterenol in different concentrations and adrenergic blocking agents on glucose induced insulin secretion and glucose utilization was studied in isolated islets of Langerhans from mice. Epinephrine in physiological concentrations significantly inhibited the glucose induced insulin secretion. This effect was not mediated by a change in glucose utilization but involved alpha-adrenergic stimulation. Isoproterenol significantly stimulated the glucose induced insulin secretion but had no effect on glucose utilization. Beta-adrenergic stimulation by isoproterenol at low glucose concentration was not sufficient to stimulate insulin secretion. The results are discussed in relation to current therories on the mechanism of glucose induced insulin secretion.     

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.