A link between insulin resistance and hyperinsulinemia: inhibitors of phosphatidylinositol 3-kinase augment glucose-induced insulin secretion from islets of lean, but not obese, rats

Wortmannin (5-100 nM), a specific phosphatidyinositol 3-kinase inhibitor, augmented 8 mM glucose-induced insulin secretion from control Sprague Dawley rat islets in a dose-dependent manner. This effect persisted after its removal from the perifusion medium; however, this augmenting effect was reduced by the calcium channel inhibitor nitrendipine or by lowering the glucose level to 3 mM. Wortmannin amplified insulin release induced by the combination of 6-8 mM glucose plus 1 microM carbachol; however, it had no effect on phorbol ester- or alpha-ketoisocaproate-induced insulin secretion. The potentiating action of wortmannin on 8 mM glucose-induced release was duplicated by LY294002. Wortmannin had no effect on glucose usage rates or inositol phosphate accumulation in [3H]inositol-prelabeled islets. Of particular significance, although 50 nM wortmannin potentiated 8 mM glucose-induced secretion from islets of lean Zucker control rats, the fungal metabolite had little effect on 8 mM glucose-induced release from islets of insulin-resistant Zucker fatty rats. These findings support the concept that the same biochemical process, inhibition ofphosphatidyinositol 3-kinase, that causes peripheral tissue insulin resistance enhances beta-cell sensitivity to glucose and produces a compensatory increase in insulin secretion from these cells. The efficacy of wortmannin depends on the in vivo status of the donor's insulin signaling pathways. This elegant biochemical control mechanism in beta-cells ensures the maintenance of glucose homeostasis despite a reduction in insulin action on peripheral tissues.     

Functional differences between aggregated and dispersed insulin-producing cells

Aims/hypothesis

Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets.

Methods

MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured.

Results

Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca²⁺]_c response.

Conclusions/interpretation

We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca²⁺]_c.     

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.     

Glucosamine-induced desensitization of beta-cell responses: possible involvement of impaired information flow in the phosphoinositide cycle.

The influence of glucosamine on beta-cell response characteristics of collagenase-isolated rat islets was determined. Groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide (PI) pools. Also included in some experiments was glucosamine (0.1-10 mM). Subsequently, these islets were perifused, and their responses to 10 mM glucose, 10 mM alpha-ketoisocaproate (KIC), and 1 microM of the phorbol ester phorbol 12-myristate 13-acetate were assessed. Increases in PI hydrolysis were monitored during the perfusion by measuring fractional efflux rates of [3H]inositol. The accumulation of inositol phosphates after the perifusion was also determined. In other experiments, the use of 10 mM glucose was measured after a 2-h exposure to 5 or 10 mM glucosamine. Finally, the ability of glucosamine itself to augment release and activate PI hydrolysis was assessed. The following observations were made. 1) A prior 2-h exposure to 5-10 mM glucosamine resulted in parallel dose-dependent impairments in 10 mM glucose-induced insulin release and PI hydrolysis. 2) Glucosamine (5-10 mM) also impaired the subsequent response to alpha-ketoisocaproate (KIC). Parallel deficits in KIC-induced PI hydrolysis were noted under conditions where insulin secretion was impaired. 3) Under several conditions where glucosamine impaired glucose-induced secretion, it had no adverse effect on phorbol 12-myristate 13-acetate-induced release. 4) The desensitizing effect of 10 mM glucosamine on 10 mM glucose-induced release and PI hydrolysis developed within 30 min of exposure to it. 5) Glucosamine (5-10 mM) preexposure had no adverse effect on the use of 10 mM glucose by desensitized islets. 6) Short term (5-min) exposure to glucosamine (10 mM) alone stimulated PI hydrolysis, while a 30-min exposure to the same level of the hexosamine depressed it. 7) In the presence of 0.25 microM forskolin, 10 mM glucosamine also had a transient stimulatory effect on insulin release. These findings support the concept that the acute and chronic effects of glucosamine on the beta-cell result at least in part from its ability to influence PI hydrolysis in islets.     

Effects of protein kinase C inhibitors on insulin secretory responses from rodent pancreatic islets

The contribution of protein kinase C (PKC) to the regulation of insulin release from perifused islets was explored using staurosporine or G? 6976 to inhibit the enzyme. Phorbol 12-myristate 13-acetate (PMA, 500 nM) addition to rat islets resulted in a slowly rising insulin secretory response. While minimally effective alone, the addition of 500 nM forskolin together with PMA resulted in a synergistic secretory response. The conventional protein-kinase-C isoform inhibitor G? 6976 (1 microM) completely abolished PMA-induced secretion. However, the combination of forskolin plus PMA significantly enhanced secretion from G? 6976-treated islets. Similar to previous findings made with staurosporine, G? 6976 (1 microM) enhanced the first phase and reduced the second phase of 20 mM glucose-induced secretion from rat islets. Additional studies were conducted comparing the secretory responses of perifused rat or mouse islets to glucose. Dramatic species differences to the hexose were observed. For example, 35-40 min after the onset of stimulation with 8, 10 or 20 mM glucose insulin release rates from mouse islets averaged 32+/-6, 84+/-27 or 131+/-17 pg/islet per minute, respectively. The responses from rat islets averaged 115+/-28, 561+/-112 or 800+/-46 pg/islet per minute at this time point. Islet insulin stores were comparable in both species. The addition of 5 microM carbachol, 500 nM forskolin or 20 mM KCl to mouse islets together with 20 mM glucose resulted in a dramatic augmentation of insulin output. The responses to carbachol or forskolin, but not KCl, were inhibited by 50 nM staurosporine. However, staurosporine (50 nM) reduced insulin secretion from rat islets stimulated with KCl plus 20 mM glucose. G? 6976 potentiated 20 mM glucose-induced secretion from mouse islets. These studies demonstrate that 1 microM G? 6976 completely abolishes PMA-induced release from rat islets and has a modest inhibitory effect on 20 mM glucose-induced secretion. G? 6976 (1 microM) had no inhibitory effect on 20 mM glucose-induced release from mouse islets. These studies also confirm that staurosporine inhibits both PKC- and PKA-mediated events in islets and this lack of specificity may account for its more pronounced inhibition of release when compared to G? 6976. Finally, significant species differences to PKC inhibitors exist between mouse and rat islets.     

The phosphoinositide 3-kinase inhibitor LY294002 enhances cardiac myocyte contractility via a direct inhibition of Ik,slow currents

OBJECTIVE: Phosphoinositide 3-kinase (PI3K) is a key component in regulating myocardial growth, survival and contractility. LY294002 and wortmannin are two PI3K inhibitors used widely to establish the role of PI3K. The goal of this study was to examine the effects of acute application of LY294002 and wortmannin on cardiac myocyte contractility and underlying mechanisms. METHODS: Patch-clamp, indo-1 epifluorescence and video-edge detection techniques were used to measure outward K(+) currents, action potentials (AP), Ca(2+) transients and shortening of myocytes isolated from mouse left ventricular free wall. RESULTS: In field-stimulated myocytes, LY294002 (10 micromol/l) increased Ca(2+) transient amplitude by 23%, and cell shortening amplitude by 60% in the absence or presence of wortmannin, while wortmannin alone had no effect. LY294002 (but not wortmannin) prolonged AP duration by specifically inhibiting slowly inactivating K(+) currents (i.e., the 4-aminopyrydine-sensitive I(k,slow1) and the tetraethylammonium-sensitive I(k,slow2)), leading to an increase in sarcoplasmic reticular Ca(2+) levels. It appeared that the AP prolongation was responsible for elevated contractility since AP-clamp of myocytes with prolonged APs (recorded in LY294002-treated myocytes) induced a 29% increase in cell shortening compared with control APs, while LY294002 application did not increase contractility in voltage-clamp studies using either step or AP depolarizations. CONCLUSIONS: The putative PI3K inhibitor LY294002 increases Ca(2+) release and myocyte contractility via direct inhibition of cardiac I(k,slow) and AP prolongation, thus limiting the usefulness of this agent in the analyses of the role of PI3K in heart function.     

Insulin secretion and the morphological and metabolic characteristics of pancreatic islets of hyperthyroid ob/ob mice.

Thyroxine treatment induced experimental hyperthyroidism in ob/ob mice, inhibited glucose-induced insulin secretion from the isolated perfused ob/ob mouse pancreas, and reduced total pancreas insulin content. In contrast, glucose-induced insulin release from incubated pancreatic islets and insulin content of pancreatic islets from ob/ob mice isolated by freehand microdissection were not reduced after thyroxine treatment when expressed per microgram dry islet. Histological examination of the ob/ob mouse pancreas revealed islets without degenerative lesions of islet cells. Granularity of beta cells was well preserved. The average number of pancreatic islets was unchanged. However, the beta cell area was significantly decreased in relation to the total pancreatic parenchyma after thyroxine treatment. This implies that insulin release and content per pancreatic islet was half of that of the controls. ATP content of islets was slightly reduced. Glucose oxidation and glucose utilization by islets from treated mice were slightly increased. Thyroxine treatment of the animals did not abolish the stimulation of 45Ca2+ uptake by glucose, but it did suppress the potentiating effect of fasting on the stimulatory effect of glucose on 45Ca2+ uptake. The metabolic characteristics of islets from experimentally hyperthyroid mice are those of all hyperthyroid tissues. The results provide no evidence for the view that the effects of thyroxine treatment may be due to disturbed metabolic function or energy deprivation of pancreatic islets. Inhibition of insulin secretion from the pancreas after thyroxine administration is apparently due to a reduction in pancreas insulin content and a diminished pancreatic islet volume. Reduced pancreatic islet volume represents most probably a reduction of individual islet cell volume.     

Aromatic amino acids and pancreatic islet function: a comparison of L-tryptophan and L-5-hydroxytryptophan

L-Tryptophan (4 mM) did not affect insulin release at 3 mM glucose but strongly potentiated glucose-induced (10 mM) insulin release in microdissected ob/ob mouse islets. The effect was concentration dependent with half-maximum at about 5 mM. 10 mM L-glutamate also enhanced the effect of 10 mM D-glucose on insulin release but L-phenylalanine, L-tyrosine, L-alanine, glycine and L-glutamine did not. 0.1 mM benserazide and 0.1 mM alpha-monofluoromethyldopa did not inhibit the effect of L-tryptophan. 1 mM aminooxyacetate reduced the potentiating effect of L-tryptophan but not that of L-5-hydroxytryptophan. 10 mM indole pyruvate stimulated basal insulin release but inhibited the effect of glucose. 10 mM L-glutamine did not enhance the stimulatory effect of indole pyruvate. 10 mM L-5-hydroxytryptophan reduced the effect of 10 mM L-glutamine on glucose oxidation. L-5-Hydroxytryptophan did not influence 14CO2 production from islets preloaded with [14C]glutamine but reduced the oxidation rate when [14C]glutamine was present in the incubation medium. Both L-tryptophan and L-5-hydroxytryptophan potentiate insulin release. The underlying mechanisms probably differ but do not seem to involve transaminations. The effect of L-5-hydroxytryptophan may be coupled to the activity of aromatic L-amino acid decarboxylase.     

Long-term leptin treatment of ob/ob mice improves glucose-induced insulin secretion.

OBJECTIVE: Previous studies have demonstrated that leptin inhibits glucose-stimulated insulin secretion from isolated islets, although a lack of leptin effect on insulin secretion has also been reported. The effect of long term in vivo leptin treatment of insulin secretion has, however, not been established. Therefore, in the present study, we have evaluated the effect of long term in vivo treatment of leptin on glucose-induced insulin secretion in ob/ob mice. METHODS: After 7 days' treatment of leptin (100 microg daily s.c.), insulin release was measured in isolated islets by batch incubation followed by radioimmunoassay. Glucose utilization and oxidation were measured by measuring the formation of (3)H(2)O and (14)CO(2) from [5-(3)H] and [U-(14)C] glucose, respectively. Glucose-6-phosphatase activity was measured by measuring the conversion of (14)C-glucose-6-P to (14)C-glucose. In addition, immunohistochemistry of pancreatic specimens was undertaken for study of expression of insulin, GLUT-2 and hormone-sensitive lipase (HSL). RESULTS: Leptin treatment significantly improved insulin secretion both at 5.5 mM (by 15%; P<0.05) and 16.7 mM (by 85%; P<0.001) glucose, compared to vehicle-treated controls. Furthermore, whereas leptin treatment did not affect islet insulin or DNA contents, a significant decrease in islet triglyceride content and glucose-6-phosphatase activity was observed. Moreover, the immunocytochemical data revealed an increased immunostaining for insulin, GLUT-2 and hormone-sensitive lipase (HSL) in islets from leptin-treated ob/ob mice. CONCLUSION: The results suggest that long-term leptin treatment of ob/ob mice improves glucose-stimulated insulin secretion in parallel with reduced glucose-6-phosphatase activity, increased HSL and decreased triglyceride levels in islets. These perturbations may explain the improvement of glucose-stimulated insulin secretion induced by leptin.     

褪黑素通过激活Akt信号通路缓解高糖诱发的原代心肌细胞损伤

目的 明确高糖对心肌细胞损伤的影响;揭示褪黑素（Mel）在高糖诱发乳鼠原代心室肌细胞损伤中的作用及机制。 方法 体外培养乳鼠原代心室肌细胞,分为4组:正常葡萄糖浓度组(NG)、高糖组(HG,HG=25 mmo/L)、高糖+褪黑素组（HG+Mel,HG=25 mmo/L;Mel=30 μmo/L）、高糖+褪黑素+ PI3K/Akt抑制剂组（HG+Mel,HG=25 mmo/L;Mel=30 μmo/L;LY294002=50 μmo/L）组,采用Western Blotting、RT-PCR和免疫荧光技术检测心肌细胞凋亡相关蛋白以及PI3K/p-Akt等指标,评价褪黑素对高糖诱发心肌细胞凋亡的作用及机制。 结果 与NG组比较,HG组心肌细胞Cl -Caspase3、Caspase9的mRNA及蛋白表达明显升高,伴有p-Akt的mRNA及蛋白表达降低和PI3K蛋白表达降低;与HG组相比,HG+Mel组心肌细胞Cl-Caspase3、Caspase9的mRNA及蛋白表达下调伴有p-Akt的mRNA及蛋白表达回升和PI3K蛋白表达升高;与HG+Mel组相比,HG+Mel+LY294002组心肌细胞的Cl-Caspase3、Caspase9的mRNA及蛋白表达上升伴有p-Akt蛋白表达水平显著降低和PI3K蛋白表达降低。免疫荧光的结果与Western Blotting、RT-PCR的结果趋势一致。 结论 褪黑素通过激活PI3K/Akt信号通路缓解高糖诱导的原代心肌细胞损伤。     

Abnormal B-cell function in neonatally streptozotocin-diabetic rats: insensitivity to alloxan toxicity

The apparent toxicity of alloxan was compared in nondiabetic rats and rats made diabetic by injection with streptozotocin during neonatal life (STZ). In the perfused pancreas of nondiabetic rats, 1 mM alloxan rapidly but evanescently stimulated insulin secretion; this effect was followed by pronounced inhibition of the insulin response to 27 mM glucose (94% inhibition) or 1 mM 3-isobutyl-1-methylxanthine (76% inhibition). Conversely, in STZ-diabetic rats the stimulatory effect of alloxan was reduced to 22% of that elicited in nondiabetic rats. In further contrast, the inhibitory effect of alloxan exposure was abolished with regard to subsequent glucose-induced insulin secretion and attenuated with regard to 3-isobutyl-1-methylxanthine-induced insulin secretion. A relative insensitivity to alloxan was also seen in collagenase-isolated islets, where alloxan completely abolished glucose-induced insulin secretion in islets from nondiabetic rats, but only nonsignificantly reduced secretion (by 37%) in islets from STZ-diabetic rats. Insensitivity to glucose in STZ diabetic rats is associated with insensitivity to alloxan. This implies a common defect in the initial recognition site of glucose and alloxan.     

Glucose affects in vitro maturation of fetal rat islets

Fetal pancreatic islets (21.5 days old) were cultured in RPMI 1640 containing either 2.8 or 11.1 mM glucose for 7 days. After the 7-day culture period, islets cultured in 2.8 mM glucose demonstrated a minimal first phase of insulin secretion in response to acute glucose stimulation, whereas islets cultured in 11.1 mM glucose demonstrated a biphasic insulin secretory pattern. Islets cultured in 11.1 mM glucose initiated insulin secretion at 4.4 +/- 0.1 mM glucose and plateaued at 11.6 mM glucose when exposed to a linear gradient. In addition, culture in 11.1 mM glucose increased DNA content (P less than 0.01) and [3H]thymidine incorporation (P less than 0.05) in fetal islets. However, ultrastructural morphometric analysis indicated that the actual number of beta-cells within islets cultured in either 2.8 or 11.1 mM glucose did not increase. The insulin contents of islets cultured in 2.8 and 11.1 mM glucose were 0.46 +/- 0.06 and 1.14 +/- 0.10 mU/islet, respectively. During subsequent glucose stimulation, islets cultured in 2.8 and 11.1 mM glucose released 3% and 5.6% of their total insulin content, respectively. Ultrastructural morphometric analysis indicated that 11.1 mM glucose stimulated an increase in the volume of individual beta-cells, i.e. hypertrophy. The hypertrophy of beta-cells within islets cultured in 11.1 mM glucose resulted in a concomitant increase in islet volume. Finally, the hypertrophy of beta-cells within islets cultured in 11.1 mM glucose was a result of increased volumes of mitochondria, secretory granules, and, to the greatest extent, endoplasmic reticulum. These findings indicate that glucose is a potent factor in the maturation of cultured fetal rat islets.     

Somatostatin inhibits oxidative respiration in pancreatic beta-cells

Somatostatin potently inhibits insulin secretion from pancreatic beta-cells. It does so via activation of ATP-sensitive K+-channels (KATP) and G protein-regulated inwardly rectifying K+-channels, which act to decrease voltage-gated Ca2+-influx, a process central to exocytosis. Because KATP channels, and indeed insulin secretion, is controlled by glucose oxidation, we investigated whether somatostatin inhibits insulin secretion by direct effects on glucose metabolism. Oxidative metabolism in beta-cells was monitored by measuring changes in the O2 consumption (DeltaO2) of isolated mouse islets and MIN6 cells, a murine-derived beta-cell line. In both models, glucose-stimulated DeltaO2, an effect closely associated with inhibition of KATP channel activity and induction of electrical activity (r > 0.98). At 100 nm, somatostatin abolished glucose-stimulated DeltaO2 in mouse islets (n = 5, P < 0.05) and inhibited it by 80 +/- 28% (n = 17, P < 0.01) in MIN6 cells. Removal of extracellular Ca2+, 5 mm Co2+, or 20 microm nifedipine, conditions that inhibit voltage-gated Ca2+ influx, did not mimic but either blocked or reduced the effect of the peptide on DeltaO2. The nutrient secretagogues, methylpyruvate (10 mm) and alpha-ketoisocaproate (20 mm), also stimulated DeltaO2, but this was unaffected by somatostatin. Somatostatin also reversed glucose-induced hyperpolarization of the mitochondrial membrane potential monitored using rhodamine-123. Application of somatostatin receptor selective agonists demonstrated that the peptide worked through activation of the type 5 somatostatin receptor. In conclusion, somatostatin inhibits glucose metabolism in murine beta-cells by an unidentified Ca2+-dependent mechanism. This represents a new signaling pathway by which somatostatin can inhibit cellular functions regulated by glucose metabolism.     

The Pancreatic β-Cell Recognition of Insulin Secretagogues: Does Cyclic AMP Mediate the Effect of Glucose?

Insulin release and the content of cAMP were studied in microdissected pancreatic islets of noninbred ob/ob (obese) mice. In the absence of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, 20 mM glucose had no effect on cAMP save a very small initial rise detectable by a freeze-stop perifusion technique only. However, combined with this methylxanthine, 20 mM glucose produced significant increases of cAMP both in perifused islets and in islets conventionally incubated in closed vials. Glucose shared this capacity to raise the cAMP level with D-glyceraldehyde and 1,3-dihydroxyacetone. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin, an activator of adenylate cyclase, also increased the islet cAMP level; the effects of the methylxanthine, whether or not combined with cholera toxin, were potentiated by glucose. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin potentiated insulin release in response to 20 mM glucose. However, only 0.5-1.0 mM isobutylmethylxanthine stimulated insulin release in the presence of 3 mM glucose, whereas 0.05-0.1 mM isobutylmethylxanthine or 5 mug/ml of cholera toxin had no effect on secretion at the low glucose concentration. These discrepancies between cAMP-promoting and insulin-releasing activities suggest that glucose does not initiate insulin release by activating the beta-cell adenylate cyclase. By being metabolized in the beta-cells, glucose may both create a release-initiating signal not identical with cAMP and enhance cAMP formation, leading to potentiation of the effect of the initiator signal.     

Glucose controls cytosolic Ca2+ and insulin secretion in mouse islets lacking adenosine triphosphate-sensitive K+ channels owing to a knockout of the pore-forming subunit Kir6.2

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (K ATP) channel-dependent Ca2+ influx with a subsequent increase of the cytosolic free Ca2+ concentration ([Ca2+]c) (triggering pathway) and a K ATP channel-independent augmentation of secretion without further increase of [Ca2+]c (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking K ATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca2+]c and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca2+]c and insulin secretion were marginally elevated, and high glucose increased [Ca2+]c only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mm KCl). Culture in 10 mm glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca2+]c with modulation of [Ca2+]c oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca2+]c and insulin secretion, by inducing K ATP channel-independent depolarization and Ca2+ influx via voltage-dependent Ca2+ channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca2+]c, but culture reveals a glucose-responsive phenotype mainly by increasing [Ca2+]c. The results support models implicating a K ATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that K ATP channels are not the only possible transducers of metabolic effects on the triggering Ca2+ signal.     

Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus

Previously we demonstrated the expression of the long form of the leptin receptor in rodent pancreatic beta-cells and an inhibition of insulin secretion by leptin via activation of ATP-sensitive potassium channels. Here we examine pancreatic islets isolated from pancreata of human donors for their responses to leptin. The presence of leptin receptors on islet beta-cells was demonstrated by double fluorescence confocal microscopy after binding of a fluorescent derivative of human leptin (Cy3-leptin). Leptin (6.25 nM) suppressed insulin secretion of normal islets by 20% at 5.6 mM glucose. Intracellular calcium responses to 16.7 mM glucose were rapidly reduced by leptin. Proinsulin messenger ribonucleic acid expression in islets was inhibited by leptin at 11.1 mM, but not at 5.6 mM glucose. Leptin also reduced proinsulin messenger ribonucleic acid levels that were increased in islets by treatment with 10 nM glucagon-like peptide-1 in the presence of either 5.6 or 11.1 mM glucose. These findings demonstrate direct suppressive effects of leptin on insulin-producing beta-cells in human islets at the levels of both stimulus-secretion coupling and gene expression. The findings also further indicate the existence of an adipoinsular axis in humans in which insulin stimulates leptin production in adipocytes and leptin inhibits the production of insulin in beta-cells. We suggest that dysregulation of the adipoinsular axis in obese individuals due to defective leptin reception by beta-cells may result in chronic hyperinsulinemia and may contribute to the pathogenesis of adipogenic diabetes.     

Differential mitogenic signaling in insulin receptor-deficient fetal pancreatic beta-cells

Insulin receptor (IR) may play an essential role in the development of beta-cell mass in the mouse pancreas. To further define the function of this signaling system in beta-cell development, we generated IR-deficient beta-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing beta-cell lines (beta-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (beta-IR(-/-)). beta-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In beta-IRLoxP beta-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S(6)K phosphorylation and beta-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S(6)K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S(6)K signaling and mitogenesis. Interestingly, in beta-IR(-/-) beta-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S(6)K phosphorylation and beta-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S(6)K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of beta-IR(-/-) cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic beta-cell lines. However, in the absence of the insulin receptor, glucose induces beta-cell mitogenesis.     

Gastrin releasing peptide augments glucose mediated 45Ca2+ uptake, electrical activity, and insulin secretion of mouse pancreatic islets

Gastrin releasing peptide (GRP) has recently been shown to increase glucose-induced insulin secretion in vivo. Being present in pancreatic tissue, the 27-amino acid peptide could play a role in the control of the glucose-induced insulin secretion of islets of Langerhans. In the presence of a stimulatory glucose concentration, GRP augmented insulin secretion of isolated islets in batch incubations. The peptide did not affect 56Rb+ efflux in the presence of 3 or 5.6 mM glucose but reduced the increase of 86Rb+ efflux evoked by the calcium ionophore A23187. 45Ca2+ uptake and intracellular recorded electrical activity induced by glucose were amplified by GRP. It is suggested that GRP plays a role in the regulation of glucose-induced insulin secretion by increasing the uptake of Ca2+ directly or by inhibition of the Ca(2+)-dependent K+ channel activity and reduced repolarization of the cell.     

Defective early phase insulin release in perifused isolated pancreatic islets of spiny mice (acomys cahirinus)

In order to characterize pancreatic beta cell function in Geneva bred spiny mice (acomys cahirinus), the dynamics of immunoreactive insulin release were examined during perifusion of pancreatic islets isolated from normoglycemic acomys. The initial insulin response of acomys was slow: no clear-cut early (1 to 10 min) peak of insulin release was observed when glucose in the perifusion medium was abruptly raised from 2.8 mM to concentrations as high as 56 mM. This was true for islets of either young, or older more obese acomys. However, after 20 to 30 min of perifusion at the high glucose concentrations, the rate of insulin release from acomysislets became similar to that from islets of rats or mice. By contrast, glucose-induced insulin release responses observed with islets of Wistar-derived rats, Swiss albino mice, and inbred C57BL/6J lean or obese (ob/ob) mice, were clearly biphasic. Tolbutamide 1.5 mM, arginine 16 mM, and theophylline 10 mM were ineffective in stimulating insulin release from acomys islets in the presence of a substimulatory glucose concentration (2.8 mM), whereas these agents were effective in rat islets at the same substimulatory concentration of glucose. On the other hand, when these agents, as well as cyclic AMP 10 mM or cytochalasin B 10 mug/ml were applied in the presence of a stimulating concentration of glucose (16.8 mM), the glucose-stimulated insulin release from acomys islets was increased to the same or to a greater extent than from rat islets. It is suggested that the failure of all the agents tested to stimulate an early rapid phase of insulin release from acomys islets may be secondary to the observed initial insensitivity to glucose, which insensitivity may in turn reflect a selective impairment in the recognition of glucose as an insulinogenic signal in this species.     

Effects of prior 5-hydroxytryptamine exposure on rat islet insulin secretory and phospholipase C responses

Glucose-induced insulin secretion is inhibited by 5-hydroxytryptamine (5HT). In the present studies the specificity of 5HT inhibition of release and the potential biochemical mechanisms involved were investigated. Dose-dependent inhibition of 15 mM glucose-induced secretion was induced by a prior 3 h incubation with 5HT. At the highest 5HT concentration (500 microM) employed, both first and second phase responses to 15 mM glucose were reduced 50-60%. In addition, this level (500 microM) of 5HT virtually abolished 10 mM glucose-induced secretion. In contrast, secretion in response to the protein kinase C activator phorbol 12-myristate 13-acetate (500 nM) was immune to 500 microM 5HT pre-treatment. Glucose usage rates were comparable in both control and 500 microM 5HT-pretreated islets. However, the generation of inositol phosphates and the efflux of 3H-inositol from 3H-inositol-prelabeled islets in response to stimulatory glucose were impaired in parallel with insulin secretion. Based on these observations the following conclusions were reached: (1) 5HT impairs glucose-induced insulin release by altering glucose-induced activation of phospholipase C. (2) Biochemical events distal to phospholipase C remain intact despite this proximal biochemical lesion. (3) Amperometric analysis of 5HT release from 5HT-pretreated islets must take into consideration its profound adverse impact on glucose-induced insulin secretion.